Complement-resistant non-mammalian DNA viruses and uses thereof

ABSTRACT

Disclosed are methods, nucleic acids, and cells for expressing an exogenous gene in a mammalian cell, involving (i) introducing into the cell a complement-resistant non-mammalian DNA virus (e.g., a baculovirus), optionally having an altered coat protein, the genome of which virus carries an exogenous gene, and (ii) growing the cell under conditions such that the gene is expressed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/927,317,filed Sep. 11, 1997 which claims priority under 35 U.S.C. §119 from U.S.Ser. No. 60/026,297 filed Sep. 11, 1996.

BACKGROUND OF THE INVENTION

This invention relates to complement-resistant non-mammalian DNA virusesand uses thereof.

Current methods for expressing an exogenous gene in a mammalian cellinclude the use of mammalian viral vectors, such as those that arederived from retroviruses, adenoviruses, herpes viruses, vacciniaviruses, polio viruses, or adeno-associated viruses. Other methods ofexpressing an exogenous gene in a mammalian cell include directinjection of DNA, the use of ligand-DNA conjugates, the use ofadenovirus-ligand-DNA conjugates, calcium phosphate precipitation, andmethods that utilize a liposome- or polycation-DNA complex. In somecases, the liposome- or polycation-DNA complex is able to target theexogenous gene to a specific type of tissue, such as liver tissue.

Typically, viruses that are used to express desired genes areconstructed by removing unwanted characteristics from a virus that isknown to infect, and replicate in, a mammalian cell. For example, thegenes encoding viral structural proteins and proteins involved in viralreplication often are removed to create a defective virus, and atherapeutic gene is then added. This principle has been used to creategene therapy vectors from many types of animal viruses such asretroviruses, adenoviruses, and herpes viruses. This method has alsobeen applied to Sindbis virus, an RNA virus that normally infectsmosquitoes but which can replicate in humans, causing a rash and anarthritis syndrome.

Non-mammalian viruses have been used to express exogenous genes innon-mammalian cells. For example, viruses of the family Baculoviridae(commonly referred to as baculoviruses) have been used to expressexogenous genes in insect cells. One of the most studied baculovirusesis Autographa californica multiple nuclear polyhedrosis virus (AcMNPV).Although some species of baculoviruses that infect crustacea have beendescribed (Blissard, et al., 1990, Ann. Rev. Entomology 35:127), thenormal host range of the baculovirus AcMNPV is limited to the orderlepidoptera. Baculoviruses have been reported to enter mammalian cells(Volkman and Goldsmith, 1983, Appl. and Environ. Microbiol.45:1085-1093; Carbonell and Miller, 1987, Appl. and Environ. Microbiol.53:1412-1417; Brusca et al., 1986, Intervirology 26:207-222; and Tjia etal., 1983, Virology 125:107-117). Although an early report ofbaculovirus-mediated gene expression in mammalian cells appeared, theauthors later attributed the apparent reporter gene activity to thereporter gene product being carried into the cell after a prolongedincubation of the cell with the virus (Carbonell et al., 1985, J. Virol.56:153-160; and Carbonell and Miller, 1987, Appl. and Environ.Microbiol. 53:1412-1417). These authors reported that, when theexogenous gene gains access to the cell as part of the baculovirusgenome, the exogenous gene is not expressed de novo. Subsequent studieshave demonstrated baculovirus-mediated gene expression in mammaliancells (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. 93:2348-2352). Inaddition to the Baculoviridae, other families of viruses naturallymultiply only in non-mammalian cells; some of these viruses are listedin Table 1.

Gene therapy methods are currently being investigated for theirusefulness in treating a variety of disorders. Most gene therapy methodsinvolve supplying an exogenous gene to overcome a deficiency in theexpression of a gene in the patient. Other gene therapy methods aredesigned to counteract the effects of a disease. Still other genetherapy methods involve supplying an antisense nucleic acid (e.g., RNA)to inhibit expression of a gene of the host cell (e.g., an oncogene) orexpression of a gene from a pathogen (e.g., a virus).

Certain gene therapy methods are being examined for their ability tocorrect inborn errors of the urea cycle, for example (see, e.g., Wilsonet al., 1992, J. Biol. Chem. 267: 11483-11489). The urea cycle is thepredominant metabolic pathway by which nitrogen wastes are eliminatedfrom the body. The steps of the urea cycle are primarily limited to theliver, with the first two steps occurring within hepatic mitochondria.In the first step, carbamoyl phosphate is synthesized in a reaction thatis catalyzed by carbamoyl phosphate synthetase I (CPS-I). In the secondstep, citrulline in formed in a reaction catalyzed by ornithinetranscarbamylase (OTC). Citrulline then is transported to the cytoplasmand condensed with aspartate into arginosuccinate by arginosuccinatesynthetase (AS). In the next step, arginosuccinate lyase (ASL) cleavesarginosuccinate to produce arginine and fumarate. In the last step ofthe cycle, arginase converts arginine into ornithine and urea.

A deficiency in any of the five enzymes involved in the urea cycle hassignificant pathological effects, such as lethargy, poor feeding, mentalretardation, coma, or death within the neonatal period (see, e.g., Emeryet al., 1990, In: Principles and Practice of Medical Genetics, ChurchillLivingstone, N.Y.). OTC deficiency usually manifests as a lethalhyperammonemic coma within the neonatal period. A deficiency in ASresults in citrullinemia which is characterized by high levels ofcitrulline in the blood. The absence of ASL results in arginosuccinicaciduria (ASA), which results in a variety of conditions includingsevere neonatal hyperammonemia and mild mental retardation. An absenceof arginase results in hyperarginemia which can manifest as progressivespasticity and mental retardation during early childhood. Othercurrently used therapies for hepatic disorders include dietaryrestrictions; liver transplantation; and administration of argininefreebase, sodium benzoate, and/or sodium phenylacetate.

The Complement System: The complement system is a group of plasmaproteins that normally helps to protect mammals from invading viral andbacterial pathogens. In the classical complement pathway, the formationof immune complexes between antibodies and antigen leads to sequentialactivation of complement factors, ultimately forming a membrane attackcomplex (MAC). The MAC forms a transmembrane channel in the target,leading to its disruption by osmotic lysis. For example, murineretroviruses are lysed by human serum after reaction with an antibody togalα(1-3)gal epitopes present on the viral envelope. Complement can alsobe activated by foreign surfaces in an alternative pathway, which doesnot require specific antibodies. Thus, complement plays a role innon-specific immune defenses which require no previous exposure to thepathogen, as well as in specific immune defenses which requireantibodies.

SUMMARY OF THE INVENTION

Disclosed herein are methods for producing a non-mammalian DNA viruscarrying an exogenous gene expression construct and having increasedresistance to complement (i.e., a “complement-resistant” virus). Ingeneral, the complement-resistant viruses of the invention are producedby propagating the virus under conditions that result in a virusparticle having a viral coat protein containing complexoligosaccharides. Such complement-resistant viruses can be used toexpress the exogenous gene in a mammalian cell, and are particularlyuseful for intravenous administration to a mammal containing a cell inwhich expression of the exogenous gene is desired. Optionally, such acomplement-resistant virus may also have an “altered” coat protein,which can be used to increase the efficiency with which thenon-mammalian DNA virus expresses the exogenous gene in the mammaliancell. For example, expression of vesicular stomatitis virus glycoproteinG (VSV-G) as an altered coat protein on the surface of a virus particleof a baculovirus enhances the ability of the baculovirus to express anexogenous gene (e.g., a therapeutic gene) in a mammalian cell.

Accordingly, the invention features a method for producing acomplement-resistant non-mammalian DNA virus by (i) introducing into anEstigmene acrea cell (e.g., an Ea4 cell or a BTI-EaA E₁ acrea cell) agenome of a non-mammalian DNA virus selected from the group consistingof baculoviruses, entomopox viruses, and densonucleosis viruses, whereinthe genome includes an exogenous gene operably linked to amammalian-active promoter; and (ii) allowing the virus to replicate inthe Estigmene acrea cell, thereby producing a complement-resistantnon-mammalian DNA virus.

The invention also features a method for producing acomplement-resistant non-mammalian DNA virus, which includes (A)providing a non-mammalian cell that expresses one or both of (i) amammalian siayltransferase and (ii) a mammalian galactosyltransferase;(B) introducing into the cell a non-mammalian DNA virus, wherein thegenome of the virus includes an exogenous gene operably linked to amammalian-active promoter; and (C) allowing the virus to replicate inthe non-mammalian cell, thereby producing a complement-resistantnon-mammalian DNA virus.

In a related method, a nucleic acid sequence encoding siayltransferaseand/or galactosyltransferase is contained within the viral genome inlieu of, or in addition to, expressing siayltransferase and/orgalactosyltransferase from the host cell. In this case, the nucleic acidsequence encoding siayltransferase or galactosyltransferase is operablylinked to a promoter that is active in the non-mammalian cell.

Another way of producing a complement-resistant non-mammalian DNA virusentails introducing into a non-mammalian cell a genome of anon-mammalian DNA virus, wherein the genome of the virus includes anexogenous gene operably linked to a mammalian-active promoter; culturingthe non-mammalian cell in a culture medium that includes one or both of(i) D-mannosamine and (ii) N-acetyl-D-mannosamine; and allowing thevirus to replicate in the non-mammalian cell, thereby producing acomplement-resistant non-mammalian DNA virus.

A related method for producing a complement-resistant non-mammalian DNAvirus entails providing a non-mammalian cell that expresses one or bothof (i) a CD59, or a homolog thereof and (ii) a decay accelerating factor(DAF), or a homolog thereof; introducing into the cell a non-mammalianDNA virus, wherein the genome of the virus includes an exogenous geneunder the control of a mammalian-active promoter; and allowing the virusto replicate in the non-mammalian cell, thereby producing acomplement-resistant non-mammalian DNA virus. Alternatively, anucleotide sequence encoding CD59, or a homolog thereof, and/or DAF, ora homolog thereof, can be contained within the viral genome in lieu of,or in addition to, expressing CD59 (or a homolog thereof) and/or DAF (ora homolog thereof) from the host cell. In this case, the nucleic acidsequence encoding CD59, DAF, and/or a homolog thereof is operably linkedto a promoter that is active in the non-mammalian cell.

In various embodiments, the genome of the non-mammalian DNA virus mayalso include a nucleic acid sequence encoding an altered coat protein.If desired, the non-mammalian cell in which the virus is propagated canbe cultured in a cell culture medium (e.g., Grace's medium or HinksTNM-FH medium) that includes one or both of (i) D-mannosamine and (ii)N-acetyl-D-mannosamine while the virus is allowed to replicate in thenon-mammalian cell, as a further method for increasing the resistance ofthe virus to complement. A variety of non-mammalian cells (e.g., insectcells) are suitable for producing complement-resistant non-mammalian DNAviruses of the invention, such as Ea4 cells, BTI-EaA E₁ acrea,Spodoptera frugiperda cells (e.g., Sf9 and Sf21), Mamestra brassicaecells, and Trichoplusia ni cells (e.g., BTI-TN-5B1-4 cells and BTI-TnMcells). Examples of suitable siayltransferases include α-2,6siayltransferase, α-2,3 siayltransferase, and α-2,8 siayltransferase. Anexemplary galactosyltransferase is β-1,4 galactosyltransferase. For geneexpression in the non-mammalian host cell, examples of suitablepromoters that can be operably linked to a nucleic acid sequence to beexpressed include the baculoviral IE1, IE2, polyhedrin, GP64, p10, andp39 promoters; Drosophila heat shock and alcohol dehydrogenasepromoters, and cytomegalovirus IE1 promoter.

As described below, the complement-resistant non-mammalian DNA virusesof the invention can be introduced into a mammalian cell, or into amammal, and the exogenous gene can be expressed in the mammalian cell orin a cell of the mammal.

The above-described methods can be used to produce a non-mammalian DNAvirus (e.g., baculovirus, entomopox virus, or densonucleosis virus)wherein the genome of the virus includes an exogenous gene operablylinked to a mammalian-active promoter, and the virus has a coat proteinthat includes a mannose core region linked to a carbohydrate moietyselected from the group consisting of N-acetyl glucosamine, galactose,and neuraminic acid.

Various cells also are included within the invention. For example, theinvention includes an Estigmena acrea cell that includes a genome of anon-mammalian DNA virus selected from the group consisting ofbaculoviruses, entomopox viruses, and densonucleosis viruses, whereinthe genome includes an exogenous gene under the control of amammalian-active promoter.

Also included is a non-mammalian cell that includes (i) a genome of anon-mammalian DNA virus, wherein the genome of the virus includes anexogenous gene under the control of a mammalian-active promoter and (ii)one or both of (a) a nucleic acid sequence encoding a mammaliansiayltransferase and (b) a nucleic acid sequence encoding a mammaliangalactosyltransferase.

Likewise, the invention features a cell culture that includes (i) anon-mammalian cell containing a genome of a non-mammalian DNA virus,wherein the genome of the virus includes an exogenous gene operablylinked to a mammalian promoter; and (ii) cell culture media thatincludes one or both of (a) D-mannosamine and (b)N-acetyl-D-mannosamine.

Also included within the invention is a nucleic acid that includes agenome of a non-mammalian DNA virus, wherein the genome of the virusincludes (i) an exogenous gene under the control of a mammalian-activepromoter and (ii) one or both of (a) a nucleic acid sequence encoding amammalian siayltransferase and (b) a nucleic acid sequence encoding amammalian galactosyltransferase. A cell containing such a nucleic acidalso is within the invention.

In a related aspect, the invention features a nucleic acid that includes(i) a genome of a non-mammalian DNA virus, wherein the genome of thevirus includes an exogenous gene under the control of a mammalian-activepromoter and (ii) one or both of (a) a nucleic acid sequence encodingCD59 or a homolog thereof and (b) a nucleic acid sequence encoding decayaccelerating factor S or a homolog thereof. A cell containing such anucleic acid also is within the invention.

The complement-resistant non-mammalian DNA viruses described herein canbe used in a variety of methods that are included within the invention.Thus, the invention also features a method of expressing an exogenousgene in a mammalian cell(s), involving (i) introducing into the cell acomplement-resistant non-mammalian DNA virus, the genome of which viruscarries the exogenous gene under the control of a promoter that inducesexpression of the exogenous gene in the cell, and (ii) maintaining thecell under conditions such that the exogenous gene is expressed.

The invention also features a method of treating a gene deficiencydisorder in a mammal (e.g., a human or a mouse), involving introducinginto a cell (in vivo or ex vivo) a therapeutically effective amount of acomplement-resistant non-mammalian DNA virus, the genome of which viruscarries an exogenous gene, and maintaining the cell under conditionssuch that the exogenous gene is expressed in the mammal.

The invention further features a method for treating a tumor in amammal, involving introducing into a cancerous cell of the mammal (e.g.,a cancerous hepatocyte) a complement-resistant non-mammalian DNA virus(e.g., a baculovirus), the genome of which virus expresses acancer-therapeutic gene (encoding, e.g., a tumor necrosis factor,thymidine kinase, diphtheria toxin chimera, or cytosine deaminase). Theexogenous gene can be expressed in a variety of cells, e.g.,hepatocytes; cells of the central nervous system, including neural cellssuch as neurons from brain, spinal cord, or peripheral nerve; adrenalmedullary cells; glial cells; skin cells; spleen cells; muscle cells;kidney cells; and bladder cells. Thus, the invention can be used totreat various cancerous or non-cancerous tumors, including carcinomas(e.g., hepatocellular carcinoma), sarcomas, gliomas, and neuromas.Included within the invention are methods for treating lung, breast, andprostate cancers. Either in vivo or in vitro methods can be used tointroduce the virus into the cell in this aspect of the invention.Preferably, the exogenous gene is operably linked to a promoter that isactive in cancerous cells, but not in other cells, of the mammal. Forexample, the α-fetoprotein promoter is active in cells of hepatocellularcarcinomas and in fetal tissue but it is otherwise not active in maturetissues. Accordingly, the use of such a promoter is preferred forexpressing a cancer-therapeutic gene for treating hepatocellularcarcinomas.

The invention also features a method for treating a neurologicaldisorder (e.g., Parkinson's Disease, Alzheimer's Disease, or disordersresulting from injuries to the central nervous system) in a mammal. Themethod involves (a) introducing into a cell a therapeutically effectiveamount of a complement-resistant non-mammalian DNA virus (e.g., abaculovirus), the genome of which virus includes an exogenous geneencoding a therapeutic protein, and (b) maintaining the cell underconditions such that the exogenous gene is expressed in the mammal.Particularly useful exogenous genes include those that encodetherapeutic proteins such as nerve growth factor, hypoxanthine guaninephosphoribosyl transferase (HGPRT), tyrosine hydroxylase,dopadecarboxylase, brain-derived neurotrophic factor, basic fibroblastgrowth factor, sonic hedgehog protein, glial derived neurotrophic factor(GDNF) and RETLI (also known as GDNFRα, GFR-1, and TRN1). Both neuronaland non-neuronal cells (e.g., fibroblasts, myoblasts, and kidney cells)are useful in this aspect of the invention. Such cells can be autologousor heterologous to the treated mammal. Preferably, the cell isautologous to the mammal, as such cells obviate concerns about graftrejection. Preferably, the cell is a primary cell, such as a primaryneuronal cell or a primary myoblast.

In each aspect of the invention, the non-mammalian DNA virus ispreferably an “invertebrate virus” (i.e., a virus that infects, andreplicates in, an invertebrate). For example, the DNA viruses listed inTable 1 can be used in the invention. Typically, the virus is a“nuclear” virus, meaning that the virus normally replicates in thenucleus, rather than cytosol, of a cell. Preferably, the invertebrateDNA virus is a baculovirus, e.g., a nuclear polyhedrosis virus, such asan Autographa californica multiple nuclear polyhedrosis virus. Ifdesired, the nuclear polyhedrosis virus may be engineered such that itlacks a functional polyhedrin gene. Either or both the occluded form andbudded form of virus (e.g., baculovirus) can be used. Other exemplaryviruses include entomopox viruses, densonucleosis viruses, and Bombyxmori nuclear polyhedrosis viruses (BmNPV).

TABLE 1 NON-MAMMALIAN DNA VIRUSES THAT CAN BE USED IN THE INVENTION.¹ I.FAMILY: BACULOVIRUSES BACULOVIRIDAE SUBFAMILY: OCCLUDED BACULOVIRUSESEUBACULOVIRINAE Genus: Nuclear polyhedrosis virus (NPV) Subgenus:Multiple Nucleocapsid Viruses (MNPV) Preferred Species: Autographacalifornica nuclear polyhedrosis virus (AcMNPV) Other Members:Choristoneura fumiferana MNPV (CfMNPV) Mamestra brassicae MNPV (MbMNPV)Orgyia pseudotsugata MNPV (OpMNPV) and approximately 400-500 speciesisolated from seven insect orders and Crustacea. Subgenus: SingleNucleocapsid Viruses (SNPV) Preferred Species: Bombyx mori S NuclearPolyhedrosis Virus (BmNPV) Other Members: Heliothis zea SNPV (HzSnpv)Trichoplusia ni SNPV (TnSnpv) and similar viruses isolated from seveninsect orders and Crustacea. Genus: Granulosis virus (GV) PreferredSpecies: Plodia interpunctella granulosis virus (PiGV) Other Members:Trichoplusia ni granulosis virus (TnGV) Pieris brassicae granulosisvirus (PbGV) Artogeia rapae granulosis virus (ArGV) Cydia pomonellagranulosis virus (CpGV) and similar viruses from about 50 species in theLepidoptera SUBFAMILY: NON-OCCLUDED BACULOVIRUSES NUDIBACULOVIRINAEGenus: Non-occluded baculoviruses (NOB) Preferred Species: Heliothis zeaNOB (HzNOB) Other Members: Oryctes rhinoceros virus Additional viruseshave been observed in a fungus (Strongwellsea magna), a spider, theEuropean crab (Carcinus maenas), and the blue crab (Callinectessapidus). II. FAMILY: ICOSAHEDRAL CYTOPLASMIC DEOXYRIBOVIRUSESIRIDOVIRIDAE Genus: Small iridescent Iridovirus insect virus groupPreferred Species: Chilo iridescent virus Other Members: Insectiridescent virus 1 Insect iridescent virus 2 Insect iridescent virus 6Insect iridescent virus 9 Insect iridescent virus 10 Insect iridescentvirus 16 Insect iridescent virus 17 Insect iridescent virus 18 Insectiridescent virus 19 Insect iridescent virus 20 Insect iridescent virus21 Insect iridescent virus 22 Insect iridescent virus 23 Insectiridescent virus 24 Insect iridescent virus 25 Insect iridescent virus26 Insect iridescent virus 27 Insect iridescent virus 28 Insectiridescent virus 29 Insect iridescent virus 30 Insect iridescent virus31 Insect iridescent virus 32 Genus: Large Iridescent Chloriridovirusinsect virus group Preferred Species: Mosquito iridescent virus(iridescent virus- type 3, regular strain) Other Members: Insectiridescent virus 3 Insect iridescent virus 4 Insect iridescent virus 5Insect iridescent virus 7 Insect iridescent virus 8 Insect iridescentvirus 11 Insect iridescent virus 12 Insect iridescent virus 13 Insectiridescent virus 14 Insect iridescent virus 15 Putative member:Chironomus plumosus iridescent Genus: Frog virus group RanavirusPreferred Species: Frog virus 3 (FV3) Other Members: Frog virus 1 Frogvirus 2 Frog virus 5 Frog virus 6 Frog virus 7 Frog virus 8 Frog virus 9Frog virus 10 Frog virus 11 Frog virus 12 Frog virus 13 Frog virus 14Frog virus 15 Frog virus 16 Frog virus 17 Frog virus 18 Frog virus 19Frog virus 20 Frog virus 21 Frog virus 22 Frog virus 23 Frog virus 24 L2L4 L5 LT 1 LT 2 LT 3 LT 4 T 21 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14T 15 T 16 T 17 T 18 T 19 T 20 Tadpole edema virus from newts Tadpoleedema virus from Rana catesbriana Tadpole edema virus from XenopusGenus: Lymphocystic disease virus group Lymphocystis virus PreferredSpecies: Flounder isolate (LCDV-1) Other Members: Lymphocystis diseasevirus dab isolate (LCDV-2) Putative member: Octopus vulgaris diseasevirus Genus Goldfish virus group Preferred Species: Goldfish virus 1(GFV-1) Other Members: Goldfish virus 2 (GF-2) III. FAMILY; PARVOVIRIDAEGenus Insect parvovirus group Densovirus Preferred Species: Galleriadensovirus Other Members: Junonia Densovirus Agraulis Densovirus BombyxDensovirus Aedes Densovirus Putative Members: Acheta Densovirus SimuliumDensovirus Diatraea Densovirus Euxoa Densovirus Leucorrhinia DensovirusPeriplanata Densovirus Pieris Densovirus Sibine Densovirus PC 84(parvo-like virus from the crab Carcinus mediterraneus) Hepatopancreaticparvo-like virus of penaeid shrimp IV. FAMILY: POXVIRUS GROUP POXVIRIDAESUBFAMILY: POXVIRUS OF INSECTS ENTOMOPOXVIRINAE Putative Genus:Entomopoxvirus A Poxvirus of Coleoptera Preferred Species: Poxvirus ofMelolontha Other Members: Coleoptera: Anomala cuprea Aphodius tasmaniaeDemodema boranensis Dermolepida albohirtum Figulus sublaevis Geotrupessylvaticus Putative Genus: Entomopoxvirus B Poxvirus of Lepidoptera andOrthoptera Preferred Species: Poxvirus of Amsacta moorei (Lepidoptera)Other Members: Lepidoptera: Acrobasis zelleri Choristoneura biennisChoristoneura conflicta Choristoneura diversuma Chorizagrotis auxiliarisOperophtera brumata Orthoptera: Arphia conspersa Locusta migratoriaMelanoplus sanguinipes Oedaleus senugalensis Schistocerca gregariaPutative Genus: Entomopoxvirus C Poxvirus of Diptera Preferred Species:Poxvirus of Chironomus luridus (Diptera) Other Members: Diptera: Aedesaegypti Camptochironomus tentans Chironomus attenuatus Chironomusplumosus Goeldichironomus holoprasimus V. GROUP CAULIFLOWER CAULIMOVIRUSMOSAIC VIRUS Preferred Member: Cauliflower mosaic virus (CaMV) (cabbageb, davis isolate) Other Members: Blueberry red ringspot (327) Carnationetched ring (182) Dahlia mosaic (51) Figwort mosaic Horseradish latentMirabilis mosaic Peanut chlorotic streak Soybean chlorotic mottle (331)Strawberry vein banding (219) Thistle mottle Putative Members: Aquilegianecrotic mosaic Cassava vein mosaic Cestrum virus Petunia vein clearingPlantago virus 4 Sonchus mottle VI. GROUP GEMINIVIRUS Subgroup I (i.e.,Genus) Maize streak virus Preferred Member: Maize streak virus (MSV)(133) Other Members: Chloris striate mosaic (221) Digitaria streakMiscanthus streak Wheat dwarf Putative Members: Bajra streak BromusStriate mosaic Digitaria striate mosaic Oat chlorotic stripe Paspalumstriate mosaic Subgroup II (i.e., Genus): Beet curly top virus PerferredMember: Beet curly top virus (BCTV) (210) Other Members: Tomatopseudo-curly top virus Bean summer death virus Tobacco yellow dwarfvirus Tomato leafroll virus Subgroup III (i.e., Genus): Bean goldenmosaic virus Preferred Member: Bean golden mosaic virus (BGMV) (192)Other Members: Abutilon mosaic virus African cassava mosaic virus Cottonleaf crumple virus Euphorbia mosaic virus Horsegram yellow mosaic virusIndian cassava mosaic virus Jatropha mosaic virus Limabean golden mosaicvirus Malvaceous chlorosis virus Melon leaf curl virus Mungbean yellowmosaic virus Potato yellow mosaic virus Rhynochosia mosaic virus Squashleaf curl virus Tigre disease virus Tobacco leaf curl virus Tomatogolden mosaic virus Tomato leaf curl virus Tomato yellow dwarf virusTomato yellow leaf curl virus Tomato yellow mosaic virus Watermeloncurly mottle virus Watermelon chlorotic stunt virus Honeysuckle yellowvein mosaic virus Putative Members: Cotton leaf curl virus Cowpea goldenmosaic virus Eggplant yellow mosaic virus Eupatorium yellow vein virusLupin leaf curl virus Soyabean crinkle leaf virus Solanum apical leafcurl virus Wissadula mosaic virus VII. FAMILY: DSDNA ALGAL VIRUSESPHYCODNAVIRIDAE Genus: dsdna Phycovirus Phycodnavirus group PreferredSpecies: Paramecium bursaria chlorella virus - 1 (PBCV-1) Viruses of:Paramecium bursaria Chlorella NC64A viruses (NC64A viruses) Parameciumbursaria Chlorella pbi viruses (pbi viruses) Hydra virdis Chlorellaviruses (HVCV) Other Members: Chlorella NC64A viruses (thirty-sevenNC64A viruses, including PBCV-1) Chlorella virus NE-8D (CV-NE8D; synonymNE-8D) CV-Nyb1 CV-CA4B CV-AL1A CV-NY2C CV-NC1D CV-BC1C CV-CA1A CV-CA2ACV-IL2A CV-IL2B CV-IL3A CV-IL3D CV-SC1A CV-SC1B CV-NC1A CV-NE8A CV-AL2CCV-MA1E CV-NY2F CV-CA1D CV-NC1B CV-NYs1 CV-IL5-2s1 CV-AL2A CV-MA1DCV-NY2B CV-CA4A CV-NY2A CV-XZ3A CV-SH6A CV-BJ2C CV-XZ6E CV-XZ4C CV-XZ5CCV-XZ4A Chlorella pbi viruses CVA-1 CVB-1 CVG-1 CVM-1 CVR-1 Hydraviridis Chlorella viruses HVCV-1 HVCV-2 HVCV-3 VIII. FAMILY:POLYDNAVIRUS POLYDNAVIRIDAE Genus: Ichnovirus Preferred Species:Campoletis sonorensis virus (CsV) Other Member: Viruses of Glypta sp.Genus: Bracovirus Preferred Species: Cotesia melanoscela virus (CmV)

If desired, the genome of the non-mammalian DNA virus can be engineeredto include one or more genetic elements selected based on their abilityto facilitate expression of (i) an altered coat protein on the surfaceof a virus particle, and/or (ii) an exogenous gene in a mammalian cell.

Any transmembrane protein that binds to a target mammalian cell, or thatmediates membrane fusion to allow escape from endosomes, can be used asthe altered coat protein on the non-mammalian DNA virus. Preferably, thealtered coat protein is the polypeptide (preferably a glycosylatedversion) of a glycoprotein that naturally mediates viral infection of amammalian cell (e.g., a coat protein of a mammalian virus, such as alentivirus, and influenza virus, a hepatitis virus, or a rhabdovirus).Other useful altered coat proteins include proteins that bind to areceptor on a mammalian cell and stimulate endocytosis. Examples ofsuitable altered coat proteins include, but are not limited to, the coatproteins listed in Table 2, which are derived from viruses such as HIV,influenza viruses, rhabdoviruses, and human respiratory viruses. Anexemplary vesicular stomatitis virus glycoprotein G (VSV-G) is encodedby the plasmid BV-CZPG, the nucleotide sequence of which is shown inFIG. 23. If desired, more than one coat protein can be used as alteredcoat proteins. For example, a first altered coat protein may be atransmembrane protein that binds to a mammalian cell, and a second coatprotein may mediate membrane fusion and escape from endosomes.

TABLE 2. EXAMPLES OF SUITABLE ALTERED COAT PROTEINS Viral Coat ProteinReference Vesicular Stomatitis Virus GenBank glycoprotein G Accession #M21416^(a) Herpes Simplex Virus 1 (KOS) GenBank glycoprotein B Accession# K01760 Human Immunodeficiency Virus GenBank type 1 gp120 Accession #U47783 Influenza A Virus GenBank hemagglutinin Accession # U38242 HumanRespiratory Syncytial GenBank Virus membrane glycoprotein Accession #M86651 Human Respiratory Syncytial GenBank Virus fusion proteinAccession #D00334 Tick-Borne Encephalitis Virus GenBank glycoprotein EAccession # S72426 Pseudorabies Virus GenBank glycoprotein gH Accession# M61196 Rabies Virus G5803FX GenBank glycoprotein Accession # U11753Human Rhinovirus 1B viral GenBank coat proteins VP1, VP2, and Accession# D00239 VP3 Semliki Forest Virus coat GenBank proteins E1, E2, and E3Accession # Z48163 Human Immunodeficiency Virus- Mebatsion et al., 1996,1 envelope spike protein PNAS 93:11366-11370 Herpes Simplex Virus-1Entry Montgomery et al., 1996, Mediator Cell 87:427-436 PseudorabiesVirus Enquist et al., 1994, Glycoprotein gE J. Virol. 68:5275-5279Herpes Simplex Virus Norais et al., 1996, Glycoprotein gB J. Virol.70:7379-7387 Bovine Syncytial Virus Renshaw et al., 1991, EnvelopeProtein Gene 105:179-184 Human Foamy Virus (HFV) GenBank Accession #Y07725 Rabies Virus glycoprotein G Gaudin et al., 1996, J. Virol.70:7371-7378 ^(a)The GenBank accession numbers refer to nucleic acidsequences encoding the viral coat proteins.

In a preferred embodiment, the altered coat protein is produced as afusion (i.e., chimeric) protein. A particularly useful fusion proteinincludes (i) a transmembrane polypeptide (e.g., antibodies such as IgM,IgG, and single chain antibodies) fused to (ii) a polypeptide that bindsto a mammalian cell (e.g., VCAM, NCAM, integrins, and selectins) or to agrowth factor. Included among the suitable transmembrane polypeptidesare various coat proteins that naturally exist on the surface of anon-mammalian or mammalian virus particle (e.g., baculovirus gp64,influenza hemagglutinin protein, and Vesicular stomatitis virusglycoprotein G). All or a portion of the transmembrane polypeptide canbe used, provided that the polypeptide spans the membrane of the virusparticle, such that the polypeptide is anchored in the membrane.Non-viral transmembrane polypeptides also can be used. For example, amembrane-bound receptor can be fused to a polypeptide that binds amammalian cell and used as the altered coat protein. Preferably, thefusion protein includes a viral coat protein (e.g., gp64) and atargeting molecule (e.g., VSV-G). Fusion polypeptides that include allor a cell-binding portion of a cell adhesion molecule also are includedwithin the invention (e.g, a gp64-VCAM fusion protein).

Typically, when the virus is engineered to express an altered coatprotein, the nucleic acid encoding the altered coat protein is operablylinked to a promoter that is not active in the mammalian cell to beinfected with the virus but is active in a non-mammalian cell used topropagate the virus (i.e., a “non-mammalian-active” promoter). Bycontrast, a mammalian-active promoter is used to drive expression of theexogenous gene of interest (e.g., a therapeutic gene), as is discussedbelow. Generally, promoters derived from viruses that replicate innon-mammalian cells, but which do not replicate in mammalian cells, areuseful as non-mammalian active promoters. For example, when using abaculovirus as the non-mammalian DNA virus, a baculovirus polyhedrinpromoter can be used to drive expression of sequence encoding thealtered coat protein, since baculoviruses do not replicate in mammaliancells. Other examples of suitable non-mammalian active promoters includep10 promoters, p35 promoters, etl promoters, and gp64 promoters, all ofwhich are active in baculoviruses. When insect cells are used to preparea virus stock, this non-mammalian-active promoter allows the alteredcoat protein to be expressed on the surface of the resulting virusparticles. Upon infecting a mammalian cell with the non-mammalian DNAvirus having an altered coat protein, the polyhedrin promoter isinactive. Examples of suitable non-mammalian-active promoters fordriving expression of altered coat proteins include baculoviralpolyhedrin promoters (e.g., from pAcAb4 from Pharmingen, Inc.), p10promoters (e.g., from pAcAb4 from Pharmingen, Inc.), p39 promoters (seeXu et al., 1995, J. Virol. 69:2912-2917), gp64 promoters (includingTATA-independent promoters; see Kogan et al., 1995, J. Virol.69:1452-1461), baculoviral IE1 or IE2 promoters (see Jarvis et al.,1996, Prot. Expr. Purif. 8:191-203), and Drosophila alcoholdehydrogenase promoters (see Heberlein et al., 1995, Cell 41:965-977)and heat shock promoters.

If desired, the non-mammalian-active promoter that is operably linked tothe gene encoding the altered coat protein can be an inducible promoterthat is activated in the non-mammalian cell in which the virus ispropagated. Examples of suitable inducible promoters include promotersbased on progesterone receptor mutants (Wang et al., 1994, Proc. Natl.Acad. Sci. 91:8180-8184), tetracycline-inducible promoters (Gossen etal., 1995, Science 268:1766-1760; 1992, Proc. Natl. Acad. Sci.89:5547-5551, available from Clontech, Inc.), rapamycin-induciblepromoters (Rivera et al., 1996, Nat. Med. 2:1028-1032), andecdysone-inducible promoters (No et al., 1996, Proc. Natl. Acad. Sci.93:3346-3351).

In principle, an inducible promoter that can be activated in either anon-mammalian or mammalian cell can be used in this embodiment of theinvention, although in practice an inducer of the promoter typicallywould be added to the non-mammalian cell in which the virus ispropagated, rather than the mammalian cell in which the exogenous geneis expressed. As an example, a gene encoding an altered coat protein canbe operably linked to a promoter that is inducible by ecdysone (No etal., 1996, Proc. Natl. Acad. Sci. 93:3346-3351). In this case, thegenome of the non-mammalian DNA virus is engineered to include a pairedecdysone response element operably linked to the gene encoding thealtered coat protein. Expression of a heterodimeric ecdysone receptor inthe presence of ecdysone (or an ecdysone analog) that is added to thecell activates gene expression from a promoter that is operably linkedto a gene encoding an altered coat protein. The use of an induciblepromoter to drive expression of the gene encoding the altered coatprotein offers the advantage of providing an additional mechanism forcontrolling expression of the altered coat protein.

The genome of the non-mammalian DNA virus can be engineered to includeadditional genetic elements, such as a mammalian-active promoter of along-terminal repeat of a transposable element or a retrovirus (e.g.,Rous Sarcoma Virus); an inverted terminal repeat of an adeno-associatedvirus and an adeno-associated rep gene; and/or a cell-immortalizingsequence, such as the SV40 T antigen or c-myc. If desired, the genome ofthe non-mammalian DNA virus can include an origin of replication thatfunctions in a mammalian cell (e.g., an Epstein Barr Virus (EBV) originof replication or a mammalian origin of replication). Examples ofmammalian origins of replication include sequences near thedihydrofolate reductase gene (Burhans et al., 1990, Cell 62:955-965),the β-globin gene (Kitsberg et al., 1993, Cell 366:588-590), theadenosine deaminase gene (Carroll et al., 1993, Mol. Cell. Biol.13:2927-2981), and other human sequences (see Krysan et al., 1989, Mol.Cell. Biol. 9:1026-1033). If desired, the origin of replication can beused in conjunction with a factor that promotes replication ofautonomous elements, such as the EBNA1 gene from EBV. The genome of thenon-mammalian DNA virus used in the invention can include apolyadenylation signal and an RNA splicing signal that functions inmammalian cells (i.e., a “mammalian RNA splicing signal), positioned forproper processing of the product of the exogenous gene. In addition, thevirus may be engineered to encode a signal sequence for proper targetingof the gene product.

The exogenous gene that is to be expressed in a mammalian cell typicallyis operably linked to a “mammalian-active” promoter (i.e., a promoterthat directs transcription in a mammalian cell), such as a “mammalian”promoter (i.e., a promoter that directs transcription in a mammaliancell, but not other cell types). Where cell-type specific expression ofthe exogenous gene is desired, the exogenous gene in the genome of thevirus can be operably linked to a mammalian-active, cell-type-specificpromoter, such as a promoter that is specific for liver cells, braincells (e.g., neuronal cells), glial cells, Schwann cells, lung cells,kidney cells, spleen cells, muscle cells, or skin cells. For example, aliver cell-specific promoter can include a promoter of a gene encodingalbumin, α-1-antitrypsin, pyruvate kinase, phosphoenol pyruvatecarboxykinase, transferrin, transthyretin, α-fetoprotein, α-fibrinogen,or β-fibrinogen. Alternatively, a hepatitis virus promoter (e.g.,hepatitis A, B, C, or D viral promoter) can be used. If desired, ahepatitis B viral enhancer may be used in conjunction with a hepatitis Bviral promoter. An albumin promoter also can be used. An α-fetoproteinpromoter is particularly useful for driving expression of an exogenousgene when the invention is used to express a gene for treating ahepatocellular carcinoma. Other preferred liver-specific promotersinclude promoters of the genes encoding the low density lipoproteinreceptor, α2-macroglobulin, α1- antichymotrypsin, α2-HS glycoprotein,haptoglobin, ceruloplasmin, plasminogen, complement proteins (C1q, C1r,C2, C3, C4, C5, C6, C8, C9, complement Factor I and Factor H), C3complement activator, β-lipoprotein, and α1-acid glycoprotein. Forexpression of an exogenous gene specifically in neuronal cells, aneuron-specific enolase promoter can be used (see Forss-Petter et al.,1990, Neuron 5: 187-197). For expression of an exogenous gene indopaminergic neurons, a tyrosine hydroxylase promoter can be used. Forexpression in pituitary cells, a pituitary-specific promoter such asPOMC may be useful (Hammer et al., 1990, Mol. Endocrinol. 4:1689-97).Typically, the promoter that is operably linked to the exogenous gene isnot identical to the promoter that is operably linked to the geneencoding an altered coat protein.

Promoters that are inducible by external stimuli also can be used fordriving expression of the exogenous gene. Such promoters provide aconvenient means for controlling expression of the exogenous gene in acell of a cell culture or within a mammal. Preferred inducible promotersinclude enkephalin promoters (e.g., the human enkephalin promoter),metallothionein promoters, mouse mammary tumor virus promoters,promoters based on progesterone receptor mutants, tetracycline-induciblepromoters, rapamycin-inducible promoters, and ecdysone-induciblepromoters. Methods for inducing gene expression from each of thesepromoters are known in the art.

Essentially any mammalian cell can be used in the invention; preferably,the mammalian cell is a human cell. The cell can be a primary cell(e.g., a primary hepatocyte, primary neuronal cell, or primary myoblast)or it may be a cell of an established cell line. It is not necessarythat the cell be capable of undergoing cell division; a terminallydifferentiated cell can be used in the invention. If desired, the viruscan be introduced into a primary cell approximately 24 hours afterplating of the primary cell to maximize the efficiency of infection.Preferably, the mammalian cell is a liver-derived cell, such as a HepG2cell, a Hep3B cell, a Huh-7 cell, an FTO2B cell, a Hepal-6 cell, or anSK-Hep-1 cell) or a Kupffer cell; a kidney cell, such as a cell of thekidney cell line 293, a PC12 cell (e.g., a differentiated PC12 cellinduced by nerve growth factor), a COS cell (e.g., a COS7 cell), or aVero cell (an African green monkey kidney cell); a neuronal cell, suchas a fetal neuronal cell, cortical pyramidal cell, mitral cell, agranule cell, or a brain cell (e.g., a cell of the cerebral cortex; anastrocyte; a glial cell; a Schwann cell); a muscle cell, such as amyoblast or myotube (e.g., a C₂C₁₂ cell); an embryonic stem cell, aspleen cell (e.g., a macrophage or lymphocyte); an epithelial cell, suchas a HeLa cell (a human cervical carcinoma epithelial line); afibroblast, such as an NIH3T3 cell; an endothelial cell; a WISH cell; anA549 cell; or a bone marrow stem cell. Other preferred mammalian cellsinclude CHO/dhfr⁻ cells, Ramos, Jurkat, HL60, and K-562 cells.

The complement-resistant virus can be introduced into a mammalian cellin vitro or in vivo. Where the virus is introduced into a cell in vitro,the infected cell can subsequently be introduced into a mammal, ifdesired. Accordingly, expression of the exogenous gene can beaccomplished by maintaining the cell in vitro, in vivo, or in vitro andin vivo, sequentially. Similarly, where the invention is used to expressan exogenous gene in more than one cell, a combination of in vitro andin vivo methods may be used to introduce the gene into more than onemammalian cell.

If desired, the virus can be introduced into the cell by administeringthe virus to a mammal that carries the cell. For example, the virus canbe administered to a mammal by subcutaneous, intravascular, orintraperitoneal injection. If desired, a slow-release device, such as animplantable pump, may be used to facilitate delivery of the virus tocells of the mammal. A particular cell type within the mammal can betargeted by modulating the amount of the virus administered to themammal and by controlling the method of delivery. For example,intravascular administration of the virus to the portal, splenic, ormesenteric veins or to the hepatic artery may be used to facilitatetargeting the virus to liver cells. In another method, the virus may beadministered to cells or an organ of a donor individual (human ornon-human) prior to transplantation of the cells or organ to arecipient.

In a preferred method of administration, the virus is administered to atissue or organ containing the targeted cells of the mammal. Suchadministration can be accomplished by injecting a solution containingthe virus into a tissue, such as skin, brain (e.g., the cerebralcortex), kidney, bladder, liver, spleen, muscle, thyroid, thymus, lung,or colon tissue. Alternatively, or in addition, administration can beaccomplished by perfusing an organ with a solution containing the virus,according to conventional perfusion protocols.

In another preferred method, the virus is administered intranasally,e.g., by applying a solution of the virus to the nasal mucosa of amammal. This method of administration can be used to facilitateretrograde transportation of the virus into the brain. This method thusprovides a means for delivering the virus to brain cells, (e.g., mitraland granule neuronal cells of the olfactory bulb) without subjecting themammal to surgery.

In an alternative method for using the virus to express an exogenousgene in the brain, the virus is delivered to the brain by osmotic shockaccording to conventional methods for inducing osmotic shock.

Where the cell is maintained under in vitro conditions, conventionaltissue culture conditions and methods may be used. In a preferredmethod, the cell is maintained on a substrate that contains collagen,such as Type I collagen or rat tail collagen, or a matrix containinglaminin. As an alternative to, or in addition to, maintaining the cellunder in vitro conditions, the cell can be maintained under in vivoconditions (e.g., in a human). Implantable versions of collagensubstrates are also suitable for maintaining the virus-infected cellsunder in vivo conditions in practicing the invention (see, e.g., Hubbellet al., 1995, Bio/Technology 13:565-576 and Langer and Vacanti, 1993,Science 260: 920-925).

The invention can be used to express a variety of exogenous genesencoding gene products such as a polypeptides or proteins, antisenseRNAs, and catalytic RNAs. If desired, the gene product (e.g., protein orRNA) can be purified from the mammalian cell. Thus, the invention can beused in the manufacture of a wide variety of proteins that are useful inthe fields of biology and medicine.

Where the invention is used to express an antisense RNA, the preferredantisense RNA is complementary to a nucleic acid (e.g., an mRNA) of apathogen of the mammalian cell (e.g., a virus, a bacterium, or afungus). For example, the invention can be used in a method of treatinga hepatitis viral infection by expressing an antisense RNA thathybridizes to an mRNA of an essential hepatitis virus gene product(e.g., a polymerase mRNA). Other preferred antisense RNAs include thosethat are complementary to a naturally-occurring gene in the cell, whichgene is expressed at an undesirably high level. For example, anantisense RNA can be designed to inhibit expression of an oncogene in amammalian cell. Similarly, the virus can be used to express a catalyticRNA (i.e., a ribozyme) that inhibits expression of a target gene in thecell by hydrolyzing an mRNA encoding the targeted gene product.Antisense RNAs and catalytic RNAs can be designed by employingconventional criteria.

If desired, the invention can be used to express a dominant negativemutant in a mammalian cell. For example, viral assembly in a cell can beinhibited or prevented by expressing in that cell a dominant negativemutant of a viral capsid protein (see, e.g., Scaglioni et al., 1994,Virology 205:112-120; Scaglioni et al., 1996, Hepatology 24:1010-1017;and Scaglioni et al., 1997, J. Virol. 71:345-353).

The invention can be used to express any of various “therapeutic” genesin a cell. A “therapeutic” gene is one that, when expressed, confers abeneficial effect on the cell or tissue in which it is present, or on amammal in which the gene is expressed. Examples of “beneficial effects”include amelioration of a sign or symptom of a condition or disease,prevention or inhibition of a condition or disease, or conferral of adesirable characteristic. Included among the therapeutic genes are thosegenes that correct a gene deficiency disorder in a cell or mammal. Forexample, carbamoyl synthetase I can correct a gene deficiency disorderwhen it is expressed in a cell that previously failed to express, orexpressed insufficient levels of, carbamoyl synthetase I. “Correction”of a gene deficiency disorder need not be equivalent to curing a patientsuffering from a disorder. All that is required is conferral of abeneficial effect, including even temporary amelioration of signs orsymptoms of the disorder. Also included are genes that are expressed inone cell, yet which confer a beneficial effect on a second cell. Forexample, a gene encoding insulin can be expressed in a pancreatic cell,from which the insulin is then secreted to exert an effect on othercells of the mammal. Other therapeutic genes include sequences thatencode antisense RNAs nucleic acid that inhibit transcription ortranslation of a gene that is expressed at an undesirably high level.For example, an antisense gene that inhibits expression of a geneencoding an oncogenic protein is considered a therapeutic gene. “Cancertherapeutic” genes are those genes that confer a beneficial effect on acancerous cell or a mammal suffering from cancer. Particularly usefulcancer therapeutic genes include the p53 gene, a herpes simplex virusthymidine kinase gene, and an antisense gene that is complementary to anoncogene.

The invention can be used to express a therapeutic gene in order totreat a gene deficiency disorder. Particularly appropriate genes forexpression include those genes that are thought to be expressed at aless than normal level in the target cells of the subject mammal.Particularly useful gene products include carbamoyl synthetase I,ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinatelyase, and arginase. Other desirable gene products includefumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor,porphobilinogen deaminase, factor VIII, factor IX, cystathioneβ-synthase, branched chain ketoacid decarboxylase, albumin,isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonylCoA mutase, glutaryl CoA dehydrogenase, insulin, β-glucosidase, pyruvatecarboxylase, hepatic phosphorylase, phosphorylase kinase, glycinedecarboxylase (also referred to as P-protein), H-protein, T-protein,Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, and CFTR (e.g., for treating cysticfibrosis).

The invention can also be used to express in a mammalian cell a genethat is expected to have a biological effect in mammals but not ininsects (i.e., a “mammal-specific” gene). For example, a baculovirusgenome can be used to express a mammalian myoD gene and thereby producemuscle proteins; such a gene would be expected to have a biologicaleffect in mammalian cells but not insect cells. Other examples ofmammal-specific genes include, but are not limited to, transcriptionfactors that function in mammalian, but not insect, cells. For example,the transcription factors c/ebp-alpha and chop10 will activate livercell differentiation pathways when expressed from an insect genome(e.g., a baculovirus genome) in a mammalian cell. In contrast,expression of these mammal-specific transcription factors in an insectcell would be expected to have a minimal, or no, effect on the insectcell.

If desired, the nucleic acids described herein can be used to propagategenetic constructs in non-mammalian (e.g., insect) cells, with theadvantage of inhibiting DNA methylation of the product. It has beenobserved that a promoter may become methylated in cell lines or tissuesin which it is not normally expressed, and that such methylation isinhibitory to proper tissue specific expression (Okuse et al., 1997,Brain Res. Mol. Brain Res. 46:197-207; Kudo et al., 1995, J. Biol. Chem.270:13298-13302). For example, a neural promoter may become methylatedin a non-neural mammalian cell. By using, for example, insect cells(e.g., Sf9 cells) to propagate a baculovirus carrying an exogenous geneand a mammalian promoter (e.g., a neural promoter), the inventionprovides a means for inhibiting DNA methylation of the promoter prior toadministration of the baculovirus and exogenous gene to the mammaliancell in which the exogenous gene will be expressed (e.g., a neuralcell).

Definitions

By “non-mammalian” DNA virus is meant a virus that has a DNA genome(rather than RNA) and which is naturally incapable of replicating in amammalian cell. Included are insect viruses (e.g., baculoviruses),amphibian viruses, plant viruses, and fungal viruses. Viruses thatnaturally replicate in prokaryotes are excluded from this definition.Examples of viruses that are useful in practicing the invention arelisted in Table 1. As used herein, a “genome” can include all or some ofthe nucleic acid sequences present in a naturally-occurringnon-mammalian DNA virus. If desired, genes or sequences can be removedfrom the virus genome or disabled (e.g., by mutagenesis), provided thatthe virus retains, or is engineered to retain, its ability to express anexogenous gene in a mammalian cell. For example, the virus can beengineered such that it lacks a functional polyhedrin gene. Such a viruscan be produced by deleting all or a portion of the polyhedrin gene froma virus genome (e.g., a baculovirus genome) or by introducing mutations(e.g., a frameshift mutation) into the polyhedrin gene so that theactivity of the gene product is inhibited.

A “complement-resistant” non-mammalian DNA virus is a non-mammalian DNAvirus that has been propagated or engineered such that it has increasedresistance to complement, relative to the wild-type non-mammalian DNAvirus. As described herein, such complement-resistant viruses can bepropagated by methods such as (i) growth on Ea cells, (ii) growth oncells expressing a mammalian siayltransferase, a mammaliangalactosyltransferase, or CD59 and/or DAF (or homologs thereof), (iii)engineering the virus to express a mammalian siayltransferase, amammalian galactosyltransferase, or CD59 and/or DAF (or homologsthereof), or (iv) by growth in a medium containing D-mannosamine and/orN-acetyl-D-mannosamine. The resulting virus can, for example, have ahybrid or complex type N-glycan coat protein (e.g., with a mannose corelinked to N-acetyl glucosamine, galactose, and/or neuraminic acid).

By “insect” DNA virus is meant a virus that has a DNA genome and whichis naturally capable of replicating in an insect cell (e.g.,Baculoviridae, Iridoviridae, Poxviridae, Polydnaviridae, Densoviridae,Caulimoviridae, and Phycodnaviridae).

By “exogenous” gene or promoter is meant any gene or promoter that isnot normally part of the non-mammalian DNA virus (e.g., baculovirus)genome. Such genes include those genes that normally are present in themammalian cell to be infected; also included are genes that are notnormally present in the mammalian cell to be infected (e.g., related andunrelated genes of other cells or species). As used herein, the term“exogenous gene” excludes a gene encoding an “altered coat protein.”

By “altered coat protein” is meant any polypeptide that (i) isengineered to be expressed on the surface of a virus particle, (ii) isnot naturally present on the surface of the non-mammalian DNA virus usedto infect a mammalian cell, and (iii) allows entry to a mammalian cellby binding to the cell and/or facilitating escape from the mammalianendosome into the cytosol of the cell. Typically, a gene encoding analtered coat protein is incorporated into the genome of thenon-mammalian DNA virus used in the invention. If desired, a virusgenome can be constructed such that the virus expresses a polypeptidethat binds a mammalian receptor or counterreceptor on a mammalian cell.An altered coat protein can include all or a portion of a coat proteinof a “mammalian” virus, i.e., a virus that naturally infects andreplicates in a mammalian cell (e.g., an influenza virus). If desired,the altered coat protein can be a “fusion protein,” i.e., an engineeredprotein that includes part or all of two (or more) distinct proteinsderived from one or multiple distinct sources (e.g., proteins ofdifferent species). Typically, a fusion protein used in the inventionincludes (i) a polypeptide that has a transmembrane region of atransmembrane protein (e.g., baculovirus gp64) fused to (ii) apolypeptide that binds a mammalian cell (e.g., an extracellular domainof VSV-G).

Although the term “altered” is used in reference to the coat protein(because it is altered in the sense that it is expressed on the surfaceof a virus particle on which it is not normally found), the proteinitself need not differ in sequence or structure from a wild-type versionof the protein. Thus, a wild-type transmembrane protein that binds amammalian cell can be used as the altered coat protein (e.g., awild-type influenza virus hemagglutinin protein). Indeed, wild-typeproteins are preferred. Nonetheless, non-wild-type proteins also can beused as the “altered” coat protein, provided that the non-wild-type coatprotein retains the ability to bind to a mammalian cell. Examples ofnon-wild-type proteins include truncated proteins, mutant proteins(e.g., deletion mutants), and conservative variations of transmembranepolypeptides that bind a mammalian cell.

“Conservative variation” denotes the replacement of an amino acidresidue by another, functionally similar, residue. Examples ofconservative variations include the substitution of one hydrophobicresidue, such as alanine, isoleucine, valine, leucine, or methionine,for another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic acid for asparticacid, or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid (i.e., amodified amino acid, such as Hydroxylysine) in place of an unsubstitutedparent amino acid.

By “positioned for expression” is meant that the DNA sequence thatincludes the reference gene (e.g., the exogenous gene) is positionedadjacent to a DNA sequence that directs transcription of the DNA and, ifdesired, translation of the RNA (i.e., facilitates the production of thedesired gene product).

By “promoter” is meant at least a minimal sequence sufficient to directtranscription. A “mammalian-active” promoter is one that is capable ofdirecting transcription in a mammalian cell. The term “mammalian-active”promoter includes promoters that are derived from the genome of amammal, i.e., “mammalian promoters,” and promoters of viruses that arenaturally capable of directing transcription in mammals (e.g., an MMTVpromoter). Other promoters that are useful in the invention includethose promoters that are sufficient to render promoter-dependent geneexpression controllable for cell-type specificity, cell-stagespecificity, or tissue-specificity (e.g., liver-specific promoters), andthose promoters that are “inducible” by external signals or agents(e.g., metallothionein, MMTV, and PENK promoters); such elements can belocated in the 5′ or 3′ regions of the native gene. The promotersequence can be one that does not occur in nature, so long as itfunctions in a mammalian cell. An “inducible” promoter is a promoterthat, (a) in the absence of an inducer, does not direct expression, ordirects low levels of expression, of a gene to which the induciblepromoter is operably linked; or (b) exhibits a low level of expressionin the presence of a regulating factor that, when removed, allowshigh-level expression from the promoter (e.g., the tet system). In thepresence of an inducer, an inducible promoter directs transcription atan increased level.

By “operably linked” is meant that a gene and a regulatory sequence(s)(e.g., a promoter) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

By “cell-immortalizing sequence” is meant a nucleic acid that, whenpresent in a mammalian cell, is capable of transforming the cell forprolonged inhibition of senescence. Included are SV40 T-antigen, c-myc,telomerase, and E1A.

By “antisense” nucleic acid is meant a nucleic acid molecule (i.e., RNA)that is complementary (i.e., able to hybridize) to all or a portion of atarget nucleic acid (e.g., a gene or mRNA) that encodes a polypeptide ofinterest. If desired, conventional methods can be used to produce anantisense nucleic acid that contains desirable modifications. Forexample, a phosphorothioate oligonucleotide can be used as the antisensenucleic acid in order to inhibit degradation of the antisenseoligonucleotide by nucleases in vivo. Where the antisense nucleic acidis complementary to only a portion of the target nucleic acid encodingthe polypeptide to be inhibited, the antisense nucleic acid shouldhybridize close enough to some critical portion of the target nucleicacid (e.g., in the translation control region of the non-codingsequence, or at the 5′ end of the coding sequence) such that it inhibitstranslation of a functional polypeptide (i.e., a polypeptide thatcarries out an activity that one wishes to inhibit (e.g., an enzymaticactivity)). Typically, this means that the antisense nucleic acid shouldbe complementary to a sequence that is within the 5′ half or third of atarget mRNA to which the antisense nucleic acid hybridizes. As usedherein, an “antisense gene” is a nucleic acid that is transcribed intoan antisense RNA. Typically, such an antisense gene includes all or aportion of the target nucleic acid, but the antisense gene is operablylinked to a promoter such that the orientation of the antisense gene isopposite to the orientation of the sequence in the naturally-occurringgene.

Use

The complement-resistant viruses of the invention can be used to expressan exogenous gene(s) in a mammalian cell in vitro or in vivo (e.g., aHepG2 cell). The viruses of the invention can also be usedtherapeutically. For example, the invention can be used to express in apatient a gene encoding a protein that corrects a deficiency in geneexpression. In alternative methods of therapy, the invention can be usedto express any protein, antisense RNA, or catalytic RNA in a cell. Theinvention also can be used in the manufacture of proteins to be purifiedfrom cells, such as proteins that are administered as pharmaceuticalagents (e.g., insulin).

The non-mammalian DNA viruses described herein, irrespective of whetherthey have been propagated to be complement-resistant, can also be usedto introduce a exogenous nucleic acid sequence into the genome of amammalian cell. Such a method can be used to correct a genetic defect orto introduce a mutation into a nucleic acid sequence in a cell. In thiscase, the nucleic acid sequence containing (a) the viral genome and (b)the exogenous nucleic acid sequence to be introduced into the cellshares a region of sequence homology with the genome of the cell intowhich the exogenous nucleic acid sequence is introduced. The exogenousnucleic acid sequence need not be operably linked to a mammalian-activepromoter in the virus. Once the nucleic acid sequence is introduced intothe cell, homologous recombination, mismatch repair, or gene conversionmethods can be used to introduce the exogenous nucleic acid sequenceinto the genome of the mammalian cell.

The complement-resistant non-mammalian viruses offer several advantages.By having increased resistance to complement, the viruses of theinvention provide increased viral stability in intravenous methods ofadministration to mammals. Thus, such viruses can be used to obtainincreased levels of exogenous gene expression in vivo. Viruses that arealso engineered to express an altered coat protein on the virus have afurther enhanced ability to infect and express a gene in a mammaliancell. Such a coat protein also can be used to confer cell-typespecificity on the engineered virus. For example, expression of CD4⁺ ona cell enhances the ability of a virus expressing an HIV envelope gp12Oprotein to infect such CD4⁺ cells (Mebatsion et al., 1996, Proc. Natl.Acad. Sci. 93:11366-11370).

The invention allows for de novo expression of an exogenous gene; thus,detection of the exogenous protein (e.g., β-galactosidase) in aninfected cell represents protein that was actually synthesized in theinfected cell, as opposed to protein that is carried along with thevirus aberrantly. Because the non-mammalian viruses used in theinvention are not normally pathogenic to humans and do not replicate inmammalian cells, concerns about safe handling of these viruses areminimized. Similarly, because the majority of naturally-occurring viralpromoters are not normally active in a mammalian cell, production ofundesired viral proteins is minimized. While traditional gene therapyvectors are based upon defective viruses that are propagated with helpervirus or on a packaging line, the invention employs a virus that is notdefective for growth on insect cells for purposes of virus propagation,but is intrinsically, and desirably, defective for growth on mammaliancells. Accordingly, in contrast to some mammalian virus-based genetherapy methods, the non-mammalian virus-based methods of the inventionare not likely to provoke a host immune response to proteins expressedby the virus in the mammalian cells.

The non-mammalian virus used in the invention can be propagated withcells grown in serum-free media, eliminating the risk of adventitiousinfectious agents occasionally present in the serum contaminating avirus preparation. In addition, the use of serum-free media eliminates asignificant expense faced by users of mammalian viruses. Certainnon-mammalian viruses, such as baculoviruses, can be grown to a hightiter (i.e., 10⁸ pfu/ml). Generally, the large virus genomes that can beused in the invention (e.g., the baculovirus genome at 130 kbp) canaccept large exogenous DNA molecules (e.g., 100 kb). In certainembodiments, the invention employs a virus the genome of which has beenengineered to contain an exogenous origin of replication (e.g., the EBVoriP). The presence of such sequences on the virus genome allowsepisomal replication of the virus, increasing persistence in the cell.Where the invention is used in the manufacture of proteins to bepurified from the cell, the invention offers the advantage that itemploys a mammalian expression system. Accordingly, one can expectproper post-translational processing and modification (e.g.,glycosylation) of the product of the exogenous gene.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the AcMNPV RSV-lacZ transferplasmid pZ4.

FIG. 2 is a schematic representation of the occluded AcMNPV RSV-lacZtransfer plasmid pZ5.

FIG. 3 is a schematic representation of the episomal transfer plasmidpZ-EBV#1, a chimera of baculovirus and Epstein Barr Virus sequences. Avirus produced with this transfer plasmid is capable of replicating in amammalian cell.

FIG. 4A is a schematic representation of a transfer plasmid that allowsexcision of a gene cassette.

FIG. 4B is a schematic representation of the gene cassette excised bythe transfer plasmid of FIG. 4A. Excision of the gene cassette ismediated by cre-lox recombination. This strategy allows persistence ofan exogenous gene in the absence of viral sequences.

FIG. 5 is a schematic representation of the transfer plasmid, pBV-AVneo,a chimera of baculovirus and Adeno-associated virus sequences. Thisplasmid is capable of integrating into the genome of the infected cell.

FIG. 6 is a schematic representation of the AcMNPV transfer plasmidpCMV-BV.

FIG. 7 is a schematic representation of the AcMNPV transfer plasmidpCMVZ-BV.

FIG. 8 is a schematic representation of the AcMNPV transfer plasmidpAct-BV.

FIG. 9 is a schematic representation of the AcMNPV transfer plasmidpAZ-BV.

FIG. 10 is a schematic representation of the AcMNPV transfer plasmidpIE45-BV.

FIG. 11 is a schematic representation of the AcMNPV transfer plasmidpNSE4-BV.

FIG. 12 is a schematic representation of the AcMNPV transfer plasmidpTH/SV40/BP9.

FIG. 13 is a schematic representation of the AcMNPV transfer plasmidpTH-Lac/BP9.

FIGS. 14A-D are photographs of cells that were stained with X-gal oneday post-infection with an AcMNPV virus containing a RSV-lacZ cassette.Cells expressing the lacZ gene stain darkly with X-gal. FIG. 14A is aphotograph of a typical field of HepG2 cells infected at a multiplicityof infection of 15. FIG. 14B is a photograph of a typical field of HepG2cells infected at a multiplicity of infection of 125; over 25% of thecells were stained. FIG. 14C is a typical field of Sk-Hep-1 cellsinfected at a multiplicity of infection of 125, showing nopositively-stained cells. FIG. 14D is a less typical field of Sk-Hep-1cells infected at a multiplicity of infection of 125 showing apositively-stained cell. Bar=55 μm.

FIG. 15 is a photograph of cells obtained following baculovirus-mediatedgene transfer into primary cultures of rat hepatocytes. Over 70% of thecells were stained blue.

FIG. 16 is a graph displaying the dose-dependence ofbaculovirus-mediated gene transfer. Here, 10⁶ HepG2 cells were seededinto 60 mm petri dishes, and one day later the cells were exposed to theindicated dose of an AcMNPV virus containing a RSV-lacZ cassette (viraltiter=1.4×10⁹ pfu/ml). At one day post-infection, the cells wereharvested, and extracts were prepared and assayed for β-galactosidaseenzyme activity. Extract activity is expressed in units ofβ-galactosidase activity as previously defined (Norton and Coffin, 1985,Mol. Cell. Biol. 5:281-290). Enzyme activity was normalized for theprotein content of each extract. Each point is the average of threeindependent assays, with the error bars representing the standarddeviation.

FIG. 17 is a graphic representation of results obtained in a time courseof baculovirus-mediated expression. HepG2 cells were infected withAcMNPV virus containing a RSV-lacZ cassette (multiplicity ofinfection=15) at time zero. After one hour, the medium containing thevirus was removed and replaced with fresh medium. Infected cells wereharvested at the indicated time points and assayed for β-galactosidaseactivity as is described above. Each plotted point is expressed as theaverage of three independent assays, with the error bars representingthe standard deviation. Expression from the virus peaked 12-24 hourspost-infection and declined thereafter when normalized to total cellularprotein.

FIG. 18 is a schematic representation of the AcMNPV transfer plasmidVSVG/BP9.

FIG. 19 is a schematic representation of the AcMNPV transfer plasmidVGZ3.

FIG. 20 is a schematic representation of a budding baculovirus having analtered coat protein. The natural baculovirus cell surface protein(gp64) and the VSV-G protein are represented by “gp64” and “VSV G.”

FIG. 21 is a schematic representation of various baculoviral transfervectors, in which an exogenous gene is operably linked to a viral ormammalian promoter.

FIG. 22 is a graphic representation of the relative transductionefficiencies of Z4 and VGZ3 in HeLa and HepG2 cells. HeLa and HepG2cells were treated with the VSV G-lacking baculovirus Z4 or the VSVG-containing baculovirus VGZ3 at multiplicities of infection of 1, 10,and 100. Expression of the lacZ gene was determined on the following dayby a in vitro chemiluminescence assay. --, HepG2 cells treated withVGZ3; -∘-, HepG2 treated with Z4; -▪-, HeLa treated with VGZ3; -□-, HeLatreated with Z4.

FIG. 23 is a listing of the nucleotide sequence of plasmid BV-CZPG,which encodes a vesicular stomatitis virus G glycoprotein.

FIG. 24 is a graph illustrating that baculoviruses propagated on Ea4cells are complement-resistant. Baculoviruses propagated on Sf21 cellswere used as a control.

FIG. 25 is a graph illustrating that baculoviruses that are (i)propagated on cells engineered to express galacatosyltransferase or (ii)engineered to express siayltransferase and propagated on cellsengineered to express galactosyltransferase are complement-resistant.Baculoviruses propagated Sf21 cells were used as a control.

DETAILED DESCRIPTION

Genetic Manipulation of Viruses

In contrast to conventional gene expression methods, the inventioninvolves modifying non-mammalian DNA viruses that do not naturallyinfect and replicate in mammalian cells. Such non-mammalian DNA virusesare further modified to render them complement-resistant by propagatingthem on particular cell types or by expressing advantageous genes fromthe viral genome. Thus, the invention is based on the addition of newproperties to a non-mammalian DNA virus that allow it to deliver a geneto a mammalian cell and direct gene expression within the mammaliancell, and which further render the virus complement-resistant. Incontrast, conventional gene therapy vectors require that viral functionsare disabled, such as expression of viral genes and viral genomereplication.

In the present method, the viral particle serves as a “shell” for thedelivery of DNA to the mammalian cell. The viral DNA is engineered tocontain transcriptional control sequences that are active in a mammaliancell, to allow expression of the gene of interest in the target cell.Conventional recombinant DNA techniques can be used for inserting suchsequences. Because the non-mammalian DNA viruses used in the inventionare not capable of replicating in mammalian cells, it is not necessaryto delete essential viral functions to render them defective. It ispreferred, however, that the virus naturally replicate in a eukaryoticspecies (e.g., an insect, a plant, or a fungus). Examples of virusesthat can be engineered to express an exogenous gene in accordance withthe invention are listed in Table 1. Preferably, the genome of the virusused in the invention is normally transported to the nucleus in itsnatural host species because nuclear localization signals functionsimilarly in invertebrate and in mammalian cells. The data summarizedbelow show that, (1) in contrast to conventional wisdom, a non-mammalianDNA virus can infect a wide variety of mammalian cells, (2) such virusescan be used to direct expression of an exogenous gene in mammaliancells, and (3) a non-mammalian DNA virus can be renderedcomplement-resistant by propagating the virus as described herein. Inaddition, expression of an altered coat protein on the surface of avirus particle enhances the ability of the virus to express an exogenousgene in a mammalian cell.

Established methods for manipulating recombinant viruses may beincorporated into these new methods for expressing an exogenous gene ina mammalian cell. For example, viral genes can be deleted from the virusand supplied in trans via packaging lines. Deletion of such genes may bedesired in order to (1) suppress expression of viral gene products thatmay provoke an immune response, (2) provide additional space in theviral vector, or (3) provide additional levels of safety in maintainingthe virus in a cell.

PROPAGATION OF VIRUSES

Complement-resistant non-mammalian DNA viruses can be propagated bymodifying conventional methods for propagating non-mammalian DNAviruses, as described below. In general, non-mammalian DNA viruses(lacking increased resistance to complement) can be propagated accordingto conventional methods as described in, e.g., Burleson, et al., 1992,Virology: A Laboratory Manual, Academic Press, Inc., San Diego, Calif.and Mahy, ed., 1985, Virology: A Practical Approach, IRL Press, Oxford,UK. Conventional conditions for propagating viruses also are suitablefor allowing expression of an altered coat protein on the surface of avirus particle. For example, baculoviruses used as controls in theexperiments described below (e.g., baculovirus not engineered to becomplement-resistant) were plaque purified and amplified according tostandard procedures (see, e.g., O'Reilly et al. infra and Summers andSmith, 1987, A Manual of Methods for Baculovirus Vectors and Insect CellCulture Procedures, Texas Agricultural Experiment Station Bulletin No.1555, College Station, Tex.). AcMNPV and Sf21 cells were propagated byspinner culture in Hinks TNM-FH media (JRH Biosciences) containing 10%fetal bovine serum (FBS) and 0.1% PLURONIC F-68™. Amplified virus can beconcentrated by ultracentrifugation in an SW28 rotor (24,000 rpm, 75minutes) with a 27% (w/v) sucrose cushion in 5 mM NaCl, 10 mM Tris pH7.5, and 10 mM EDTA. The viral pellet is then resuspended inphosphate-buffered saline (PBS) and sterilized by passage through a 0.45μm filter (Nalgene). If desired, the virus may be resuspended bysonication in a cup sonicator. AcMNPV was titered by plaque assay onSf21 insect cells.

Various methods for producing complement-resistant viruses in accordancewith the invention are described below.

1) Growth of Virus on Estigmena acrea Cells: A complement-resistantnon-mammalian DNA virus can be produced by propagating a non-mammalianDNA virus (such as a baculovirus, entomopox virus, or densonucleosisvirus) on cells derived from the salt marsh caterpillar Estigmena acrea(Ea), such as Ea4 cells (available from Novagen, Inc.; Madison, Wis.) orBTI-EaA E₁ acrea cells (Ogonah et al., 1996, Biotechnology 14:197).Methods for isolating and culturing Ea cells are known in the art (see,e.g., Ogonah et al., 1996, Nature Biotech. 14:197-202). In an exemplarymethod, and for the examples described below, Ea4 cells are cultured at27° C. in Grace's medium (supplemented) plus 10% fetal calf serum.Without being bound by any particular theory, propagation of viruses onEa4 cells is thought to result in more complex N-linked glycosylation ofviral coat proteins than does propagation of viruses on other insectcells (e.g., Sf cells), thereby rendering the virus resistant tocomplement.

2) Growth of Virus on Cells in Media Containing D-mannosamine and/orN-acetyl-D-Mannosamine: A related method for producing acomplement-resistant non-mammalian DNA virus entails propagating thevirus on non-mammalian cells grown in a medium containing D-mannosamineand/or N-acetyl-D-mannosamine. Any of a variety of host cells can beused in this method, such as Ea cells (e.g., Ea4 cells and BTI-EaA E₁acrea), Sf9 cells, Sf21 cells, Mamestra brassicae cells, andTrichoplusia ni cells (e.g., BTI-TN-5B1-4 cells (High Five™ cells);(Invitrogen, Inc.; San Diego, Calif.) or BTI TnM cells (Wickham et al.,1992, Biotechnol. Prog. 8:391-396)). Without being bound by anyparticular theory, D-mannosamine and N-acetyl-D-mannosamine are thoughtto increase the amount of sialic acid on the virus particle. BothD-mannosamine and N-acetyl-D-mannosamine are commercially available(Sigma; St. Louis, Mo.) and each can be included in the cell culturemedium at a concentration of 0.1 mM to 100 mM (e.g., 5 mM to 30 mM). Anyconventional cell culture medium for propagating the non-mammalian cellline (e.g., Grace's medium and Hinks TNM-FH medium) can be used andsupplemented with D-mannosamine and/or N-acetyl-D-mannosamine. The virusand cells then can be cultured, and the virus isolated, usingconventional procedures, for example as described above. The resultingvirus can be used to infect mammalian cells as described herein.

3) Growth of Virus on Cells Expressing Mammalian Siavltransferase and/orGalactosvltransferase: Another method for producing complement-resistantvirus entails propagating the virus on cells that have been engineeredto express a mammalian siayltransferase and/or galactosyltransferase.Examples of suitable cells include Ea, Sf9, Sf21, and Trichoplusia nicells. Suitable siayltransferase and galactosyltransferase genes havebeen isolated (see, e.g., Sjoberg et al., 1996, J. Biol. Chem.271:7450-7459, GenBank Accession No. X74570, and the TIGR Human GeneIndex THC Report TCH212460). Examples of suitable siayltransferasesinclude α-2,6 siayltransferase, α-2,3 siayltransferase and α-2,8siayltransferase. An exemplary galactosyltransferase is β-1,4galactosyltransferase (e.g., bovine β-1,4 galactosyltransferase). Thesiayltransferase and/or galactosyltransferase gene(s) can readily beexpressed in insect cells using conventional methods. For example, thegene(s) can be expressed in insect cells by using the Insect SelectSystem (Invitrogen), which uses the vector pIZ/V5-His, which contains abaculovirus (Orgyia pseudotsugata) immediate early 2 (IE2) promoter, orby expressing the gene under the control of a baculoviral vector IE1,polyhedrin, GP64, or p10 promoter, a CMV IE1 promoter, or a Drosophilaheat shock promoter.

4) Expression of Mammalian Siavltransferase and/or Galactosyltransferasefrom the Virus: In lieu of, or in addition to, expressing a mammaliansiayltransferase and/or galactosyltransferase gene on a vector or fromthe genome of the cells used for virus propagation, the non-mammalianDNA virus can be engineered to contain and express a siayltransferaseand/or galactosyltransferase gene(s) in the cells used to propagate thevirus. Conventional recombinant DNA methods can be used to engineer anon-mammalian DNA virus containing a siayltransferase and/orgalactosyltransferase gene under the control of a promoter that directsgene expression in the host cell (e.g., the baculoviral IE1, IE2, GP64,polyhedrin, and p10 promoters, the CMV IE1 promoter, or the Drosophilaheat shock promoter). Such a promoter need not be active in mammaliancells subsequently infected by the virus. Without being bound by anyparticular theory, expression of siayltransferase and/orgalactosyltransferase in the cell during virus propagation is thought toproduce a non-mammalian DNA virus having viral coat proteins withcomplex oligosaccharides, thereby rendering the virus resistant tocomplement.

5) Growth of Virus on Cells Expressing Human CD59 or DAFComplement-inhibiting Genes: In an alternative method,complement-resistant virus can be produced by propagating the virus oncells (e.g., Ea, Sf9, Sf21, or Trichoplusia ni cells) that express humanCD59 and/or decay accelerating factor (DAF) complement-inhibiting genes,or their homologs (e.g., a mammalian homolog of CD59 (such as the mousehomolog Ly-6), the complement control protein homolog encoded byherpesvirus saimiri (Fodor et al., 1995, J. Virol. 69:3889-3892), or arat homolog of human DAF (Hinchliffe et al., 1998, J. Immunol.161:5695-5703). Nucleic acids encoding human CD59 and DAF are readilyavailable (see, e.g., ATCC Nos. 65964, 65965, 379846, and 449654;GenBank Accession Nos. R67545, H54186, N36869; and Medof et al., 1987,Proc. Nat'l. Acad. Sci. 84:2007-2011) and can be expressed in the cellfrom a vector, under the control of a promoter that directs geneexpression in the host cell (e.g., the baculoviral IE1, IE2, GP64,polyhedrin, or p10 promoter, a Drosophila heat shock promoter, or a CMVIE1 promoter). If desired, a nucleic acid encoding CD59 or DAF can bestably integrated genome of the host cell used to propagate the virus.

6) Growth of Virus on Virus Expressing Human CD59 or DAFComplement-Inhibiting Genes: In lieu of, or in addition to, propagatingthe virus on a cell line expressing CD59 and/or DAF, the virus itselfcan be engineered to express CD59 and/or DAF. To this end, conventionalrecombinant DNA techniques can be used to engineer a non-mammalian DNAvirus containing the CD59 and/or DAF genes under the control of apromoter that is active in the cells used to propagate the virus (e.g.,a baculoviral IE1, IE2, GP64, polyhedrin, or p10 promoter, a Drosophilaheat shock promoter, or a CMV IE1 promoter).

ALTERED COAT PROTEINS

In various embodiments, the invention involves the expression of analtered coat protein(s) on the surface of virus particle to enhance theability of a non-mammalian DNA virus to infect a mammalian cell andexpress an exogenous gene in the mammalian cell. Conventional molecularbiology techniques and criteria can be used for identifying andexpressing on the virus a polypeptide that binds a mammalian cell.Typically, a gene encoding the altered coat protein is operably linkedto a non-mammalian-active promoter, and is expressed from the viralgenome. Alternatively, the altered coat protein can be encoded by asequence contained within a chromosome of a non-mammalian cell in whichthe virus is propagated. Upon expression of the altered coat proteinfrom the cellular chromosome, the altered coat protein is packaged alongwith the non-mammalian DNA virus. In yet another alternative method, thealtered coat protein can be expressed from the genome of a second virusthat co-infects the non-mammalian cell in which the non-mammalian DNAvirus is propagated. Thus, upon co-infection and expression of thealtered coat protein from the genome of the second virus, the alteredcoat protein is packaged along with the non-mammalian DNA virus.Regardless of the method used to express the altered coat protein, thenon-mammalian DNA virus is maintained under conditions such that thealtered coat protein is expressed on the surface of the virus particle.To this end, conventional methods for propagating viruses innon-mammalian cells can be used. If desired, expression of the alteredcoat protein on the surface of a virus particle can be confirmed usingconventional techniques, such as immunoblotting, immunofluorescence, andthe like.

Conventional molecular biology techniques can be used to produce asuitable fusion protein that is used as the altered coat protein. Forexample, where a baculovirus is used as the non-mammalian DNA virus, awide variety of fusion proteins can be made employing the baculoviruscoat protein gp64 (Whitford et al., 1989, J. Virol. 63:1393-1399 andAyres et al., 1994, Virology 202:586-605). The baculovirus expressionvector pAcSurf-2 provides a gp64 gene having a multiple cloning sitepositioned in-phase between the gp64 signal sequence and the sequenceencoding the mature glycoprotein (Boublik et al., 1995, Biotechnology13:1079-1084). Sequences encoding a polypeptide that binds a mammaliancell can readily be inserted into the multiple cloning site of thisvector, and expression of the resulting fusion protein is driven by thepolyhedrin promoter to which the gp64 sequences are operably linked.

OTHER GENETIC ELEMENTS

If desired, the viral capsid or envelope can contain, as part of thealtered coat protein, or as a separate molecule in addition to thealtered coat protein, a ligand that binds to mammalian cells tofacilitate entry. For example, the virus can include as a ligand anasialoglycoprotein that binds to mammalian lectins (e.g., the hepaticasialoglycoprotein receptor), facilitating entry into mammalian cells.

Because most promoters of non-mammalian viruses are not active inmammalian cells, the exogenous gene should be operably linked to apromoter that is capable of directing gene transcription in a mammaliancell (i.e., a “mammalian-active” promoter). Examples of suitablepromoters include the RSV LTR, the SV40 early promoter, CMV IE promoters(e.g., the human CMV IE1 promoter), the adenovirus major late promoter,and the Hepatitis viral promoters (e.g., a Hepatitis B viral promoter).Other suitable “mammalian-active” promoters include “mammalianpromoters,” i.e., sequences corresponding to promoters that naturallyoccur in, and drive gene expression in, mammalian cells. Often,“mammalian promoters” are also cell-type-specific, stage-specific, ortissue-specific in their ability to direct transcription of a gene, andsuch promoters can be used advantageously in the invention as a meansfor controlling expression of the exogenous gene. For example, severalliver-specific promoters, such as the albumin promoter/enhancer, havebeen described and can be used to achieve liver-specific expression ofthe exogenous gene (see, e.g., Shen et al., 1989, DNA 8:101-108; Tan etal., 1991, Dev. Biol. 146:24-37; McGrane et al., 1992, TIBS 17:40-44;Jones et al., J. Biol. Chem. 265:14684-14690; and Shimada et al., 1991,FEBS Letters 279:198-200). Where the invention is used to treat ahepatocellular carcinoma, an α-fetoprotein promoter is particularlyuseful. This promoter is normally active only in fetal tissue; however,it is also active in liver tumor cells (Huber et al., 1991, Proc. Natl.Acad. Sci. 88:8039-8043). Accordingly, an α-fetoprotein promoter can beused to target expression of a liver-cancer therapeutic to liver tumorcells.

If desired, the virus genome can be engineered to carry an origin ofreplication in order to facilitate persistence of the exogenous gene inthe mammalian cell. Origins of replication derived from mammalian cells(i.e., “mammalian origins of replication,” have been identified (Burhanset al., 1994, Science 263:639-640). Other origins of replication thatfunction in mammals (i.e., “mammalian-active” origins, e.g., theEpstein-Barr Virus oriP) can also facilitate maintenance of expressionin the presence of appropriate trans-acting factors (e.g., EBNA-1). Ifdesired, the virus can be engineered to express more than one exogenousgene (e.g., the virus can be engineered to express both OTC and AS) ormore than one altered coat protein.

EXAMPLES OF TRANSFER PLASMIDS

Descriptions of several viruses used in the examples described below nowfollow. These examples are provided for illustrative purposes, and arenot meant to limit the scope of invention.

Construction of the DZ4 Transfer Plasmid: Genetic manipulation of abaculovirus for use in the invention can be accomplished withcommonly-known recombination techniques originally developed forexpressing proteins in baculovirus (see, e.g., O'Reilly et al., 1992,In: Baculovirus expression vectors, W. H. Freeman, New York). In thisexample, an AcMNPV was constructed by interrupting the polyhedrin geneof the virus with a cassette that directs expression of a reporter gene.The reporter gene cassette included DNA sequences corresponding to theRous Sarcoma Virus (RSV) promoter operably linked to the E. coli lacZgene (FIG. 1). The reporter gene cassette also included sequencesencoding Simian Virus 40 (SV40) RNA splicing and polyadenylationsignals.

The RSV-lacZ AcMNPV transfer plasmid used in several examples set forthbelow is named Z4 and was constructed as follows. An 847 bp fragment ofpRSVPL9 including the SV40 RNA splicing signal and polyadenylationsignal was excised using BglII and BamHI. Plasmid pRSVPL9 was derivedfrom pRSVglobin (Gorman et al., Science 221:551-553) by digestingpRSVglobin with BglII, adding a HindIII linker, and then cleaving theDNA with HindIII. A double-stranded polylinker made by hybridization ofthe oligonucleotides 5′AGCTGTCGACTCGAGGTACCAGATCTCTAGA3′ (SEQ ID NO: 1)and 5′AGCTTCTAGAGATCTGGTACCTCGAGTCGAC3′ (SEQ ID NO: 2) was ligated tothe 4240 bp fragment having the RSV promoter and SV40 splicing andpolyadenylation signals. The resulting plasmid has the polylinker inplace of the globin sequences. The SV40 sequence of pRSVPL9 was clonedinto the BamHI site of pVL1392 (Invitrogen and Pharmingen) usingstandard techniques. The resulting intermediate plasmid was namedpVL/SV40. An RSV-lacZ cassette was excised from pRSVlacZII (Lin et al.,1991, Biotechniques 11:344-348, and 350-351) with BglII and SpeI andinserted into the BglII and XbaI sites of pVL/SV40.

The AcMNPV RSV-lacZ virus, termed Z4, was prepared by homologousrecombination of the Z4 transfer plasmid with linearized AcMNPV DNA. TheAcMNPV virus used to prepare this DNA was AcV-EPA (Hartig et al., 1992,J. Virol. Methods 38:61-70).

Construction of the DZ5 Transfer Plasmid: Certain non-mammalian viruses(e.g., baculoviruses) may be occluded in a protein inclusion body (i.e.,occluded-derived viruses (ODV)), or they may exist in a plasma membranebudded form. Where an occluded virus is used in the invention, the virusmay first be liberated from the protein inclusion body, if desired.Conventional methods employing alkali may be used to release the virus(O'Reilly et al., 1992, In: Baculovirus expression vectors, W. H.Freeman, New York). An occluded, alkali-liberated baculovirus may betaken up by a cell more readily than is the non-occluded budded virus(Volkman and Goldsmith, 1983, Appl. and Environ. Microbiol.45:1085-1093). To construct the pZ5 transfer plasmid (FIG. 2), for usingan occluded virus in the invention, the RSV-lacZ cassette was excisedfrom the pZ4 transfer plasmid using BglII and BamHI and then insertedinto the BglII site of pAcUW1 (Weyer et al., 1990, J. Gen. Virol.71:1525-1534).

Construction of the PZ-EBV#1 Transfer Plasmid: The non-mammalian DNAviruses used in the invention may be engineered to permit episomalreplication of the virus in the mammalian cell. Such a virus wouldpersist longer, thereby optimizing methods for long-term expression ofan exogenous gene in a cell. An example of such a replicating virus ispZ-EBV#1 (FIG. 3), which was constructed as follows. The EBV orip andEBNA-1 region was excised from pREP9 (Invitrogen) using EcoRI and XbaIand then inserted into the baculoviral transfer plasmid pBacPAK9(Clontech) at its EcoRI and XbaI sites, yielding pEBVBP9. The RSV-lacZcassette was excised from transfer plasmid Z4 with BglII and BamHI andthen inserted into the BamHI site of pEBVBP9 to yield the plasmidpZ-EBV#1.

Construction of PZ4loxP: The Z4loxP viral genome is a substrate forrecombination with bacteriophage P1 cre recombinase. This virus can beused to insert gene cassettes bearing a loxP site into the virus usingstandard procedures (Patel et al., 1992, Nucl. Acids Res. 20:97-104). Avariation of this insertion system may be engineered so that the viralsequences are excised from the remaining gene expression sequences. Forexample, an auto-excising transfer plasmid may be constructed (FIGS.4A-4B) to express an exogenous gene in a mammalian cell. This plasmidcontains loxP sequences which facilitate excision of the baculoviralsequences. The pZ4loxP transfer plasmid was constructed by inserting asynthetic loxP site into the pZ4 transfer plasmid. Two loxPoligonucleotides were synthesized and annealed to each other. Theoligonucleotides were:5′GATCTGACCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTAAGG3′ (SEQ ID NO: 3)and 5′GATCCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCA3′ (SEQ ID NO:4). The oligonucleotides were annealed by heating them to 80° C. in thepresence of 0.25 M NaCl and then allowing the mixture to cool slowly toroom temperature before use in the ligation reactions. The annealedoligonucleotides were then ligated to the pZ4 transfer plasmid that hadbeen digested with BglII. The ligations and analysis of the resultingclones were performed with standard cloning techniques. RecombinantZ4loxP baculovirus was then generated with conventional methods forrecombination into linear baculoviral DNA.

Construction of pBV-AVneo, an AAV Chimera Transfer Plasmid: Abaculovirus genome that is capable of integrating into a chromosome ofthe host cell can also be used in the invention. Such an integratedvirus may persist in the cell longer than a non-integrated virus.Accordingly, methods of gene expression involving such viruses mayobviate the need for repeated administration of the virus to the cell,thereby decreasing the likelihood of mounting an immune response to thevirus. The transfer plasmid pBV-AVneo (FIG. 5) includes the invertedterminal repeats of an Adeno-associated virus (AAV). This transferplasmid was constructed by excising the neo gene, which encodesG418-resistance, as a BglII-BamHI fragment from pFasV.neo and insertingthe fragment into the BamHI site of pAVgal in place of the lacZ gene.Plasmid pAVgal was constructed by replacing the rep and cap codingsequences of AAV with a CMV promoter and a lacZ gene. The resultingintermediate fragment, termed pAV.neo, was digested with PvuI. The largePvuI fragment, which has the CMV promoter driving expression of the neogene, flanked by the AAV ITRs, then was inserted into the PacI site ofpBacPAK9. If desired, a suitable promoter operably linked to an AAV repgene may be inserted into this construct (e.g., between the AAV ITR andthe polyhedrin promoter) to facilitate excision and recombination intothe genome. Examples of rep genes that may be inserted into thisconstruct include rep40, rep52, rep68, and rep78.

Construction of the pCMV-BV Transfer Plasmid: The human cytomegalovirusimmediate early promoter, a 758 bp HindIII-XbaI fragment, was excisedfrom PCMV-EBNA (Invitrogen) at HindIII, BamHI and inserted into theHindIII sites of pBluescript (SKII⁺), yielding plasmid pCMV-SKII⁺. Thepromoter was then excised from CMV-SKII⁺ at the XhoI, BamHI sites andinserted into the XhoI, BglII sites of pSV/BV, yielding plasmid pCMV-BV(FIG. 6). pSV/BV is a modified version of the baculovirus transferplasmid pBacPAK9 (Clontech), containing an altered polylinker and SV40splice and polyadenylation signals. pSV/BV was constructed byrestriction of pBacPAK9 with NotI, treatment with T4 DNA polymerase tocreate blunt ends, and self-ligation to remove the NotI site. A new NotIsite was then added by ligation of the linker pGCGGCCGC into the SmaIsite. Finally, SV40 splice and polyadenylation sequences were added bydigestion of pRSVPL with BglII-BamHI, and insertion of the 847 bpfragment into the BamHI site of the modified BacPAK9, yielding pSV/BV.

Construction of the pCMVZ-BV Transfer Plasmid: pCMVZ-BV (FIG. 7) wasconstructed by restriction of pCMV-BV with NotI and ligation insertionof a 3 kb lacZ fragment. The lacZ fragment was prepared by restrictionof pAlb-Gal with NotI.

Construction of the pAct-BV Transfer Plasmid: The 345 bp rat β-actinpromoter was excised from pINA (Morgenstern, JP, 1989, Ph.D. Thesis,University College, London, UK) at BglII, BamHI and inserted into theBglII site of pSV/BV, yielding pAct-BV (FIG. 8).

Construction of the PAZ-BV Transfer Plasmid: pAZ-BV (FIG. 9) wasconstructed by restriction of pAct-BV with NotI and ligation insertionof a 3 kb lacZ fragment. The lacZ fragment was prepared by restrictionof pAlb-Gal with NotI.

Construction of the pIE45-BV Transfer Plasmid: pIE45-BV (FIG. 10) wasconstructed by restriction of pHSVPrPUC (Neve et al., 1997, Neuroscience79:435-447) with SphI, followed by treatment with T4 DNA polymerase inthe presence of nucleotide triphosphates to create blunt ends. PstIlinkers (New England Biolabs, Catalog #1024, pGCTGCAGC) were then addedby treatment with T4 DNA ligase, the fragment of approximately 850 bpwas subjected to digestion with PstI, and cloned into the PstI site ofpSV/BV.

Construction of the pNSE4-BV Transfer Plasmid: pNSE4-BV (FIG. 11) wasconstructed by restriction of pNSE4 (see, e.g., Quon et al., 1991,Nature 352::239-241 and Forss-Petter et al., 1990, Neuron 5:187-197)with SalI and EcoRI, followed by ligation into the XhoI and EcoRI sitesof pSV/BV.

Construction of the pTH/SV40/BP9 Transfer Plasmid: pTH/SV40/BP9 (FIG.12) was constructed by restriction of pTH4.8 Thdno (Banerjee et al.,1992, J. Neuroscience 12:4460-4467) with EcoRI and NotI, and ligation ofthe 4.0 kb promoter fragment into pSV/BV, which was also digested withEcoRI and NotI.

Construction of the pTH-Lac/BP9 Transfer Plasmid: pThlac (FIG. 13) wasconstructed by restriction of pALB-Gal with Not I and isolation of the 3kb lacZ fragment, which was then ligated into pTH/SV40/BP9 which wasalso restricted with Not I using T4 DNA ligase.

EXAMPLES OF EXOGENOUS GENE EXPRESSION

Because non-mammalian DNA viruses were long thought not to be capable ofinfecting and directing gene expression in mammalian cells, Part A ofthe examples below provides evidence that non-mammalian DNA viruses(e.g., a baculovirus) can, in fact, be used to express an exogenous genein a mammalian cell. Although the examples described in Part A employviruses that have not been propagated according to the above-describedmethods for producing complement-resistant viruses, these examplesprovide support for the assertions that a complement-resistantnon-mammalian DNA virus can be used to express an exogenous gene in amammalian cell. In addition, these examples provide guidance forpracticing the invention with a complement-resistant non-mammalian DNAvirus.

The examples in Part B, below, utilize non-mammalian DNA viruses thathave an altered coat protein. Because the presence of the altered coatprotein is the only significant difference between the viruses of theinvention and the viruses that lack an altered coat protein, theseexamples demonstrate that the expression of the altered coat proteinenhances the ability of a non-mammalian DNA virus to express anexogenous gene in a mammalian cell. Accordingly, in each of the methodsdescribed below (e.g., in vivo expression of an exogenous gene), theviruses having an altered coat protein are expected to be superior tothe viruses lacking the altered coat protein.

The examples in Part C, below, demonstrate that non-mammalian DNAviruses can be propagated to provide increased resistance to complementcontained in mammalian serum. These complement-resistant viruses werepropagated according to the methods described above for producingcomplement-resistant non-mammalian DNA viruses.

Part A: A Non-Mammalian DNA Virus can be used to Express an ExogenousGene in a Mammalian Cell

I. Examples of Expression of an Exogenous Gene in Mammalian Cells InVitro

Nearly all mammalian cells are potential targets of non-mammalianviruses, and any cultured or primary cell can rapidly be tested. In thefollowing example, the ability of the Z4 baculovirus to infect 19different types of cells was tested. In this example, the baculoviruswas the Z4 virus, prepared by homologous recombination of the Z4transfer plasmid with linearized AcMNPV DNA. The tested cells wereHepG2, Sk-Hep-1, NIH3T3, NIH3T3 cells expressing a cell-surfaceasialoglycoprotein receptor, HeLa, CHO/dhfr⁻, 293, COS, Ramos, Jurkat,HL60, K-562, C₂C₁₂ myoblasts, C₂C₁₂ myotubes, primary human musclemyoblasts, Hep3B cells, FTO2B cells, Hepa1-6 cells, and nerve growthfactor-differentiated PC12 cells.

Growth of Cells: Conventional tissue culture methods can be used to growmammalian cells to be infected (Freshney, 1987, Culture of Animal Cells:A Manual of Basic Techniques, 2nd ed., Alan R. Liss, Inc. New York,N.Y.). These cells were grown and infected as is described above. Thecells were grown as follows. HepG2 and Sk-Hep-1 cells were cultured inminimal essential medium as modified by Eagle (EMEM) containing 10% FBS.NIH3T3, HeLa, 293, and COS cells were cultured in DMEM containing 10%FBS. CHO/dhfr⁻ cells were cultured in MEM alpha containing 10% FBS.Ramos, Jurkat, HL60, and K-562 cells were cultured in RPMI 1640 mediumcontaining 10% FBS. HL60 cells were induced to differentiate by culturein the same medium containing 0.5% dimethyl sulfoxide and 1 μM retinoicacid (Sigma). C₂C₁₂ myoblasts were propagated in DMEM containing 20% FBSand differentiated to myotubes during culture in DMEM containing 10%horse serum. PC12 cells were propagated in DMEM containing 5% FBS and10% horse serum, and were induced to differentiate during culture inDMEM containing 10% FBS, 5% horse serum, and 100 ng/ml nerve growthfactor. All cells were seeded one day prior to infection with AcMNPV,and multiplicities of infection were calculated assuming a doubling incell number during this time. The C₂C₁₂ and PC12 cells may haveincreased in cell number during differentiation, and therefore reflect asomewhat lower moi.

In vitro Infection of Cells: In vitro infection of mammalian cells witha virus can be accomplished by allowing the virus to adsorb onto thecells for 0.1 to 6 hours; preferably, adsorption proceeds for 1 to 2hours. Generally, a multiplicity of infection of 0.1 to 1,000 issuitable; preferably, the moi is 100 to 500. For relatively refractorycells, a moi of 100 to 1,000 is preferable. For the viruses used in theinvention, the titer may be determined with conventional methods whichemploy the non-mammalian cells that the virus naturally infects. Ifdesired, the mammalian cell to be infected may be maintained on a matrixthat contains collagen (e.g., rat tail Type I collagen). Based on cellcounting after culture and infection of cells on collagen-coated platesand comparison with cells grown on a conventional EHS matrix, I havefound that a collagen matrix increases the susceptibility of cells(e.g., liver cells) to infection by a non-mammalian virus by 10 to 100fold, relative to a conventional EHS matrix. Commercially-availableplates containing a collagen matrix are available (e.g., BIO-COAT™plates, Collaborative Research), and rat tail collagen is alsocommercially available (Sigma Chemical and Collaborative Research).

In the in vitro assays described below, standard conditions forinfection utilized 2×10⁶ cells and RSV-lacZ AcMNPV at a moi of 15.Adherent cell lines were seeded one day prior to infection. Cells wereexposed to virus in 2 ml of medium for 90 minutes, and then thevirus-containing medium was removed and replaced with fresh medium.Mock-infected cells were treated with 2 ml medium lacking the viralinoculum.

Detection of Infection and Gene Expression: Delivery of a virus to acell and expression of the exogenous gene can be monitored usingstandard techniques. For example, delivery of a virus (e.g., AcMNPV) toa cell can be measured by detecting viral DNA or RNA (e.g., by Southernor Northern blotting, slot or dot blotting, or in situ hybridization,with or without amplification by PCR). Suitable probes that hybridize tonucleic acids of the virus, regulatory sequences (e.g., the promoter),or the exogenous gene can be conveniently prepared by one skilled in theart of molecular biology. Where the invention is used to express anexogenous gene in a cell in vivo, delivery of the virus to the cell canbe detected by obtaining the cell in a biopsy. For example, where theinvention is used to express a gene in a liver cell(s), a liver biopsycan be performed, and conventional methods can be used to detect thevirus in a cell of the liver.

Expression of an exogenous gene in a cell of a mammal can also befollowed by assaying a cell or fluid (e.g., serum) obtained from themammal for RNA or protein corresponding to the gene. Detectiontechniques commonly used by molecular biologists (e.g., Northern orWestern blotting, in situ hybridization, slot or dot blotting, PCRamplification, SDS-PAGE, immunostaining, RIA, and ELISA) can be used tomeasure gene expression. If desired, a reporter gene (e.g., lacZ) can beused to measure the ability of a particular baculovirus to target geneexpression to certain tissues or cells. Examination of tissue caninvolve: (a) snap-freezing the tissue in isopentane chilled with liquidnitrogen; (b) mounting the tissue on cork using O.C.T. and freezing; (c)cutting the tissue on a cryostat into 10 μm sections; (d) drying thesections and treating them with 4% paraformaldehyde in PBS, followed byrinsing in PBS; (e) staining the tissue with X-gal (0.5mg/ml)/ferrocyanide (35 mM)/ferricyanide (35 mM) in PBS; and (f)analyzing the tissue by microscopy.

To measure expression of the reporter gene in the infected cells,calorimetric assays of β-galactosidase enzymatic activity were performedwith standard methods (Norton et al., 1985, Molecular & Cellular Biology5:281-290). Other conventional methods for measuring β-galactosidaseactivity could be used in lieu of the methods employed in this example.Cell extracts were prepared at one day post-infection. Cell monolayerswere rinsed three times with PBS, scraped from the dish, and collectedby low-speed centrifugation. The cell pellets were resuspended in 25 mMTris pH 7.4/0.1 mM EDTA and then subjected to three cycles of freezingin liquid nitrogen and thawing in a 37° C. water bath. The extracts werethen clarified by centrifugation at 14,000× g for 5 minutes. Standardconditions for assaying β-galactosidase activity utilized 0.1 ml of cellextract, 0.8 ml of PM-2 buffer, and 0.2 ml ofo-nitrophenyl-α-D-galactopyranoside (4 mg/ml) in PM-2 buffer for 10minutes at 37° C. (Norton et al., 1985, Mol. & Cell. Biol. 5:281-290).The reaction was stopped by the addition of 0.5 ml of 1 M sodiumcarbonate. The amount of substrate hydrolyzed was detectedspectrophotometrically at 420 nm, and β-galactosidase enzymatic activitywas calculated with conventional methods (Norton et al., 1985, Mol. &Cell. Biol. 5:281-290). The assay was verified to be linear with respectto extract concentration and time. Extract protein concentrations weredetermined using the Coomassie Plus protein assay (Pierce) with bovineserum albumin as a standard, and the level of β-galactosidase activitywas expressed as units of β-galactosidase activity per mg of protein.Other standard protein assays can be used, if desired.

For histochemical staining of β-galactosidase activity, cells were fixedin 2% (w/v) formaldehyde-0.2% (v/v) paraformaldehyde in PBS for 5minutes. After several rinses with PBS, the cells were stained by theaddition of 0.5 mg/ml of X-gal (BRL) in PBS for 2-4 hours at 37° C.

Assay of 19 Mammalian Cell Types: The following 19 examples illustratethat expression of an exogenous gene can be detected in 14 of the 19mammalian cell types that were tested. These assays employed twodifferent tests of β-galactosidase activity. By X-gal staining, the moresensitive assay, exogenous gene expression was detected in 14 of the 19mammalian cell types. Using an ONPG assay of cell extracts, which is aless sensitive assay, three of the cell lines (HepG2, 293, and PC12)showed statistically significant (P<0.05, Student's t-test) higherβ-galactosidase activity after exposure to the virus (Table 3). Thehuman liver tumor line HepG2 exposed to the RSV-lacZ baculovirusexpressed greater than 80-fold higher levels of β-galactosidase than didmock-infected controls. The adenovirus-transformed human embryonalkidney cell line 293 expressed the lacZ reporter gene at a level ofabout four-fold over background. In addition, PC12 cells, which weredifferentiated to a neuronal-like phenotype with nerve growth factor,exhibited about two-fold higher β-galactosidase levels after infectionwith the RSV-lacZ baculovirus. This difference was statisticallysignificant (P=0.019).

TABLE 3 BACULOVIRUS-MEDIATED EXPRESSION OF AN RSV-LACZ REPORTER GENE INMAMMALIAN CELL LINES. β-galactosidase activity (units/mg) Mean ± SD CellLine Mock Infected RSV-lacZ Virus HepG2 0.030 ± 0.004 2.628 ± 0.729Sk-Hep-1 0.019 ± 0.003 0.019 ± 0.004 NIH3T3 0.026 ± 0.003 0.023 ± 0.005HeLa 0.034 ± 0.009 0.036 ± 0.005 CHO/dhfr- 0.020 ± 0.002 0.026 ± 0.005293 0.092 ± 0.014 0.384 ± 0.024 COS 0.029 ± 0.002 0.032 ± 0.007 Ramos0.008 ± 0.002 0.011 ± 0.004 Jurkat 0.012 ± 0.004 0.007 ± 0.001 HL600.042 ± 0.039 0.014 ± 0.015 K-562 0.018 ± 0.006 0.017 ± 0.002 C₂C₁₂myoblast 0.015 ± 0.001 0.014 ± 0.003 C₂C₁₂ myotube 0.049 ± 0.011 0.042 ±0.004 PC12 (+NGF) 0.019 ± 0.005 0.033 ± 0.004

By histochemical staining, a more sensitive assay, β-galactosidaseactivity was detected in 14 of the 19 cell lines exposed to virus. Thus,certain of the cell lines that did not yield statistically significantlyhigher levels of β-galactosidase, as measured in extracts, were, infact, able to express β-galactosidase at low, but reproducible,frequencies, as detected by the more sensitive X-gal staining procedure.This frequency could be increased by using higher multiplicities ofinfection such that cells that, at a low moi appear not to express thegene, stain blue at a higher moi. Examples of cell lines that could betransfected in this manner include SK-Hep-1, NIH3T3, HeLa, CHO/dhfr⁻,293, Cos, and C₂C₁₂ cells. In addition, β-galactosidase activity wasdetected in primary human muscle myoblasts that were exposed to virus.This finding indicates that baculovirus was able to mediate genetransfer both to primary cells and the corresponding established cellline (C₂C₁₂), indicating that expression of the exogenous gene in anestablished cell line has predictive value for the results obtained withprimary cells.

β-galactosidase activity was also detected in Hep3B cells treated withthe virus; the level of expression in these cells was nearly equivalentto the level detected with HepG2 cells. In addition, β-galactosidaseactivity was found in FTO2B (rat hepatoma) cells and Hepa1-6 (humanhepatoma) cells exposed to virus. β-galactosidase activity was alsodetected in NIH3T3 cells that were engineered to express theasialoglycoprotein receptor on the cell surface. These cells expressedapproximately two times the level of β-galactosidase as did normalNIH3T3 cells. This observation suggests that an asialoglycoproteinreceptor may be used to increase susceptibility to viral-mediated genetransfer.

At the moi employed, the Ramos, Jurkat, HL60, and K-562 cell lines didnot express statistically significant levels of β-galactosidase, asrevealed by β-galactosidase enzyme assays after infection. Based on theresults with other mammalian cell lines, it is expected thatβ-galactosidase activity would be detected in these apparentlyrefractory cell lines when a higher dose (i.e., moi) of virus or longeradsorption time period is utilized.

Even when exposure of cells to the virus results in expression of theexogenous gene in a relatively low percentage of the cells (in vitro orin vivo), the invention can be used to identify or confirm the cell- ortissue-type specificity of the promoter that drives expression of theexogenous gene (e.g., a reporter gene such as a chloramphenicolacetyltransferase gene, an alkaline phosphatase gene, a luciferase gene,or a green fluorescent protein gene). Once identified, such a promotermay be employed in any of the conventional methods of gene expression.Similarly, only relatively low levels of expression are necessary forprovoking an immune response (i.e., produce antibodies) in a mammalagainst the heterologous gene product. Thus, the gene expression methodof the invention can be used in the preparation of antibodies against apreferred heterologous antigen by expressing the antigen in a cell of amammal. Such antibodies may be used inter alia to purify theheterologous antigen. The gene expression method may also be used toelicit an immunoprotective response in a mammal (i.e., be used as avaccine) against a heterologous antigen. In addition, the invention canbe used to make a permanent cell line from a cell in which the virusmediated expression of a cell-immortalizing sequence (e.g., SV40 Tantigen).

Histochemical staining using X-gal provided a highly sensitive methodfor detecting β-galactosidase expression in cells exposed to themodified AcMNPV. When HepG2 cells were exposed to the modified AcMNPV ata moi of 15, about 5-10% of the cells stained with X-gal (FIG. 14A). Ata multiplicity of infection (moi) of 125, about 25-50% of the cells werestained (FIG. 14B). No adverse effects of exposure to the virus, such asnuclear swelling, were observed. These data demonstrate that themodified AcMNPV is highly effective at gene transfer into HepG2 cellswhen a sufficient dose of virus is used. When the Sk-Hep-1 line wasexposed to virus at a moi of 15, no stained cells were observed (datanot shown). While the majority of Sk-Hep-1 cells that were exposed tovirus at a moi of 125, did not stain blue (FIG. 14C), a few cells werefound that stained darkly after treatment with this higher doses ofvirus (FIG. 14D). These data indicate that cells that appear to berefractory to the virus at a relatively low moi can, in fact, beinfected, and express the exogenous gene, at a higher moi. Stained cellswere not found in mock-infected cultures (data not shown). The frequencyof stained cells in the Sk-Hep-1 cell line was estimated to be2,000-4,000 fold less than in HepG2 cells after exposure to equivalentdoses of the modified virus, as determined by cell counting. Thus, thecell type-specificity demonstrated by the modified AcMNPV is relativerather than absolute. These data also indicate that, where a mixture ofcells is contacted with the virus (in vitro or in vivo), the dosage ofthe virus can be adjusted to target the virus to the cells that areinfected at a lower moi.

Expression in Primary Cultures of Rat Hepatocytes: This exampleillustrates that a non-mammalian DNA virus can also be used to expressan exogenous gene at high levels in primary cultures of rat hepatocytes.In this experiment, freshly prepared rat hepatocytes were plated ontodishes coated with rat tail collagen as previously described (Rana etal., 1994, Mol. Cell. Biol. 14:5858-5869). After 24 hours, the cellswere fed with fresh medium containing RSV-lacZ baculovirus at amultiplicity of infection of approximately 430. After an additional 24hours, the cells were fixed and stained with X-gal. Over 70% of thecells were stained-blue, indicating that they have taken up andexpressed the RSV-lacZ cassette (FIG. 15). The frequency of expressionobtained in this example is higher than the frequency reported withconventional viral vectors used in gene therapy (e.g., retroviral andHerpes Simplex Virus vectors). Mock-infected cultures did not containany positively-stained cells (data not shown). Other preferred exogenousgenes can be used in lieu of the lacZ gene. In addition, other primarycells can readily be plated and incubated with a non-mammalian cell inlieu of the primary rat hepatocytes.

Expression in Cortex Cultures: The following two examples illustratethat a non-mammalian DNA virus can be used to express an exogenous genein cultured neuronal and glial cells. For this example, the Z4 virus wasprepared from Sf9 cells grown in Hink's TNM-FH media containing 10% FCS,as described above. The virus was purified by banding on a 20-60%sucrose gradient in phosphate-buffered saline. The titer of the virusemployed in the following experiments was 3×10⁸ pfu/ml (for virus stock#1) or 2×10⁹ pfu/ml (for virus stock # 2), as measured on Sf9 cells.Each virus stock was sonicated prior to use.

For the first example, rat cerebral cortex cultures were prepared fromE16 embryonic pups. A 24-well dish was seeded with 300,000 cells/well,and, at 4 days post-plating, the cells were infected by adding varyingamounts of virus in serum-containing medium to the wells, as isindicated in Table 4. The virus was allowed to adsorb onto the cells for24 hours.

TABLE 4 EXPRESSION OF AN EXOGENOUS GENE IN RAT CORTICAL CELLS. Virus 1μl 2 μl 5 μl 10 μl 50 μl 100 μl Z4 Stock #1 moi = 1 moi = 2 moi = 5 moi= 10 moi = 50 moi = 100 no blue no blue ˜5 blue ˜20 blue ˜500 blue ˜2200blue cells cells cells cells cells cells (˜0.75%) Z4 Stock #2 moi = 6.7moi = 13.3 moi = 34 moi = 67 moi = 335 moi = 667 few blue ˜100 blue ˜200blue ˜450 blue ˜1000 blue ˜1300 blue cells cells cells cells cells cellsPBS no blue cells no blue cells no blue cells

Expression of the exogenous β-galactosidase gene was measured bycounting the number of blue cells after staining the cells with X-gal.Table 4 provides the number of blue cells observed in five fields of themicroscope at 10× magnification; each well contained approximately 65fields. In some wells, the cells at the periphery of the well werepreferentially stained.

These data indicate that the exogenous β-galactosidase gene wasexpressed from the virus in the cultured neuronal cells. In contrast, noblue cells were detected when the cell cultures were mock-infected withPBS. Thus, this non-mammalian virus can be used to express an exogenousgene in neuronal and glial cells, as determined by the detection of bluecells that were, by cell morphology, identified as neurons and gliaaccording to standard criteria.

In the second example, the Z4 baculovirus was used to express anexogenous gene in cultured cortical cells obtained from rat pups at theE20 and P1 stages. The cells from E20 pups were plated in 24-well dishesat 380,000 cells/well. The cells from P1 pups were plated at 300,000cells/well. The E20 cultures were treated with araC (to inhibit thegrowth of glia) at 6 days post-plating, and they were infected at 10days post-plating. The P1 cultures were treated with araC at 2 dayspost-plating, and they were infected at 6 days post-plating. Samples ofeach culture were infected with various dilutions of Z4 virus at titer2×10⁹ pfu/ml. To measure the strength of the RSV promoter, the cellswere also infected, in separate experiments, with Herpes Simplex Virus(HSV) expressing the lacZ gene under two different promoters. In onecase, cells were infected with a HSV in which the lacZ gene was placedunder the control of an RSV promoter. The titer of this HSV stock was2×10⁷ IU/ml, as measured on PC12 cells with X-gal histochemistry. Forcomparison, the cells were infected with a HSV in which the lacZ genewas placed under control of the HSV IE4/5 promoter. The titer of thisvirus was 2×10⁸ IU/ml, as measured on PC12 cells with X-galhistochemistry. For a negative control, the cells were mock-infectedwith PBS. Expression of the exogenous lacZ gene was measured by countingthe number of blue cells obtained upon staining the cells with X-gal.

The non-mammalian Z4 virus of the invention successfully expressed theexogenous lacZ gene in cultured cortical cells obtained from rat pups atboth the E20 and Pi stages of development. With 1-100 μl of the Z4virus, 4.9-10% of the cortical cells at the E20 stage, and 2.1-5.75% ofthe cortical cells at the Pi stage, were stained blue with X-gal,indicating expression of the exogenous gene in those cells. Of the cellsinfected with 0.1-5.0 μl of the HSV RSVlacZ virus, as a positivecontrol, 1.9-3.4% of the E20 cells, and 0.45-4.2% of the P1 cellsstained blue with X-gal. When the cells were infected with a 5 μl sampleof HSV expressing lacZ from the IE4/5 promoter, nearly 100% of the cellsstained blue. When E20 or P1 cortical cells were mock-infected with PBS,as a negative control, no blue cells were detected. These data provideadditional evidence that the non-mammalian Z4 baculovirus can be used toexpress an exogenous gene in cortex cells. These data also indicate thatthe level of expression obtained with the Z4 virus is comparable to thelevel of expression obtained with HSV.

Dose-response of Baculovirus-mediated Gene Transfer: The histochemicaldata presented above indicate that increasing amounts of β-galactosidaseare produced after exposure of mammalian cells to increasing amounts ofvirus. To quantitate the dose-dependence of baculovirus-mediated geneexpression, HepG2 cells were exposed to increasing doses of virus andassayed for β-galactosidase enzyme activity. The amount of enzymeproduced was linearly related to the inoculum of virus used over a widerange of doses (FIG. 16). This suggests that entry of each virusparticle occurs independently of entry of other virus particles. Themaximum dose of virus used in this assay was limited by the titer andvolume of the viral stock, and no plateau in the amount of expressionwas observed using higher doses of virus. Accordingly, these dataindicate that, in practicing the invention, one can modulate the levelexpression (i.e., the percent of cells in which the exogenous gene isexpressed) by adjusting the dosage of virus used.

Time Course of Baculovirus-mediated Gene Transfer: HepG2 cells wereexposed to the RSV-lacZ virus for 1 hour, after which the cells wereharvested at various times and quantitatively assayed forβ-galactosidase activity. As is shown in FIG. 17, β-galactosidaseactivity was detected as early as 6 hours after exposure to the virus,and expression peaked 12-24 hours post-infection. As is expected for anepisomal DNA molecule, expression from the RSV-lacZ cassette graduallysubsided at later time (FIG. 17 and data not shown). LacZ expressionremained detectable by X-gal staining at 12 days post-transfection infewer than 1 in 1,000 cells (data not shown). This expression of LacZwas not the result of viral spread, because culture supernatants takenfrom HepG2 cells 10 days post-infection had titers of 10 pfu/ml asdetermined by plaque assay on Sf21 cells. These data suggest that, wherethe invention is used in the manufacture of proteins that are purifiedfrom HepG2 cells, it may be desirable to isolate the protein from thecell at a time not sooner than 6 hours after infection of the cell.Depending on the half-life of the protein, it may be desirable toisolate the protein shortly after the peak in protein expression (i.e.,after approximately 22-26 hours (e.g., approximately 24 hours)post-infection for HepG2 cells). The optimal time period for maximizingisolating the manufactured protein can readily be determined for eachprotein, virus, and cell.

Expression Occurs De Novo in Mammalian Cells: These examples confirmthat expression of the exogenous gene occurs de novo in mammalian cells.To demonstrate that the detected reporter gene activity in the mammaliancells was not simply the result of β-galactosidase being physicallyassociated with AcMNPV virions as they enter the mammalian cell, severalexperiments were performed that demonstrate that the observed expressionof the lacZ reporter gene was the result of de novo synthesis ofβ-galactosidase. First, the RSV-lacZ virus inoculum was assayed forβ-galactosidase activity, and the level of β-galactosidase activity wasfound to be less than 10% of that expressed after infection of HepG2cells. Second, HepG2 cells were infected with the RSV-lacZ virus andthen cultured in the presence of the protein synthesis inhibitorcycloheximide. Inclusion of cycloheximide after infection inhibited theaccumulation of β-galactosidase enzyme activity by more than 90% (Table5). Third, HepG2 cells were infected at an equivalent moi with BacPAK6(Clontech), a baculovirus in which the lacZ gene was under control ofthe viral polyhedrin promoter rather than the RSV promoter (Table 5).The latter virus expresses extremely high levels of β-galactosidaseactivity in insect cells where the promoter is active (data not shown).In mammalian cells, the viral polyhedrin promoter is inactive, and thevirus containing this promoter failed to provide any enzyme activity inmammalian cells (Table 5). In contrast to prior studies of baculovirusinteractions with mammalian cells, these data demonstrate that de novosynthesis of lacZ occurs after baculovirus-mediated gene transfer into amammalian cell.

TABLE 5 BACULOVIRUS-MEDIATED GENE EXPRESSION OCCURS DE NOVO.β-galactosidase Drug During Drug Post (% of RSV-lacZ, Virus InfectionInfection mean ± SD) RSV-lacZ none none 100 ± 5.8   none none none 3.2 ±0.4 RSV-lacZ none cycloheximide 10.3 ± 1.0  BacPAK6 none none 2.8 ± 0.4RSV-lacZ chloroquine chloroquine 2.9 ± 0.1 RSV-lacZ none chloroquine25.1 ± 6.2 

Baculovirus-mediated Gene Transfer is Inhibited by Lysomotropic Agents:To gain insight into the mechanism by which baculoviruses express anexogenous gene in a mammalian cell, the susceptibility of geneexpression to a lysomotropic agent was examined. Like other envelopedviruses, the budded form of AcMNPV normally enters cells viaendocytosis, followed by low pH-triggered fusion of the viral envelopewith the endosomal membrane, thus allowing escape into the cytoplasm(Blissard et al., 1993, J. Virol. 66:6829-6835; Blissard et al., 1990,Ann. Rev. of Entomol. 35:127-155). To determine whether endosomeacidification was necessary for baculovirus-mediated gene transfer intomammalian cells, HepG2 cells were infected with RSV-lacZ AcMNPV in thepresence of chloroquine, a lysomotropic agent. HepG2 cells were exposedto AcMNPV virus in media containing or lacking inhibitor for 90 minutes,then the virus-containing media were removed and replaced with freshmedia containing or lacking inhibitors as listed.

At one day post-infection, the cells were harvested and extracts wereassayed for β-galactosidase activity and protein content. Each value inthe table represents the average of three independent assays, with theamount of β-galactosidase produced by the RSV-lacZ AcMNPV virus in theabsence of inhibitors assigned a value of 100%. β-galactosidase activitywas normalized for protein content of each extract. When 25 μMchloroquine was continuously present during and after exposure of HepG2cells to the virus, de novo expression of β-galactosidase was completelyprevented (Table 5). This suggests that baculovirus-mediated genetransfer is dependent upon endosomal acidification. When chloroquine wasadded to the cells at 90 minutes after exposure to the virus, onlypartial inhibition of β-galactosidase expression was observed.Apparently, a portion (≈22%) of the viral particles were able to proceedthrough the endosomal pathway during the 90 minutes of exposure to thevirus.

Baculovirus-mediated Gene Transfer is Enhanced by Butyrate: This exampleillustrates that butyrate enhances the ability of a baculovirus toexpress an exogenous gene in a mammalian cell. Five transfer plasmidscontaining different mammalian promoters were created, as diagrammed inFIG. 21. These vectors were constructed using pSV/BV, a modified versionof the baculovirus transfer plasmid pBacPAK9 (Clontech), containing analtered polylinker and SV40 splice and polyadenylation signals. pSV/BVwas constructed by restriction of pBacPAK9 with NotI, treatment with T4DNA polymerase to create blunt ends, and self-ligation to remove theNotI site. A new NotI site was then added by ligation of the linkerpGCGGCCGC into the SmaI site. Finally, SV40 splice and polyadenylationsequences were added by digestion of a variant of pRSVglobin withBglII-BamHI, and insertion of the 850 bp fragment into the BamHI site ofthe modified BacPAK9, yielding pSV/BV. The human cytomegalovirusimmediate early promoter, 758 bp HindIII-XbaI fragment, was excised frompCMV-EBNA (Invitrogen) at HindIII, BamHI and inserted into the HindIII,BamHI sites of pBluescript (SKII⁺), yielding plasmid pCMV-SKII⁺. Thepromoter was then excised from CMV-SK II⁺ at the XhoI, BamHI sites andinserted into the XhoI, BglII sites of pSV/BV, yielding plasmid pCMV/BV.The 500 bp mouse phosphoglycerate kinase (PGK) promoter was prepared bycutting pKJ1-neo (Tybulewicz, 1991, Cell 65: 1153-1163) with EcoRI andmade blunt with T4 DNA polymerase to remove the EcoRI site. Theresulting pKJ1 plasmid lacking the EcoRI site was amplified by pfupolymerase chain reaction using the primers5′ACCGCGGATCCAATACGACTCACTATAG3′ (SEQ ID NO: 5) and 5°CGGAGATCTGGAAGAGGAGAACAGCGCGGCAG3′ (SEQ ID NO: 6). The amplified PGKpromoter was then digested with XhoI and BglII and inserted into thesame sites of pSV/BV yielding PKJ1/BV. The 345 bp rat β-actin promoterwas excised from pINA (6) at BglII, BamHI and inserted into the BglIIsite of pSV/BV yielding pβ-actin/BV. The 2.3 kb albumin enhancer and 700bp albumin promoter were excised from pGEMAlbSVPA (Zaret et al., 1988,Proc. Natl. Acad. Sci. 85: 9076-9080) at NaeI, NsiI and inserted intothe SmaI, PstI sites of pSV/BV. The RSVlacZ transfer plasmid used (alsoreferred to herein as the Z4 virus) is described above. A 3.0 kb Lac Zcassette was inserted into the NotI site of all of the plasmidsconstructed (See FIG. 21).

Recombinant viruses were generated by contransfection of the baculovirustransfer vectors with linear BP6 viral DNA (Clontech) into Sf21 cells.The recombinant viruses were purified through three rounds of plaqueisolation and amplified on Sf21 cells. The amplified viruses wereconcentrated by ultracentrifugation as described above and titered by a96-well method on Sf21 insect cells (O'Reilly et al., 1992, BaculovirusExpression Vectors: A Laboratory Manual, W. H. Freeman, New York, N.Y.).

The human hepatocellular carcinoma cell line HepG2 was infected witheach recombinant virus at a multiplicity of infection of 100. Twomillion cells were infected in a final volume of 1 ml Eagle's MinimumEssential Medium in a 60 mm tissue culture dish. The infection wasallowed to proceed for two hours, then 4 ml of complete medium was addedto the cells. In a second series of HepG2 infections, the conditions ofthe first infections were repeated with the exception that after theinfection had proceeded for 2 hours 25 μl of sodium butyrate (100 mM)was added to the cells with 1.5 ml complete media. As a control, cellswere mock-infected to assess background β-galactosidase enzyme activity.The cell monolayers were collected after 24 hours and prepared for acalorimetric assay (with ONPG) of β-galactosidase enzymatic activity asdescribed above. Hepatocytes were isolated by collagenase perfusion andplated on rat tail collagen as previously described (Boyce et al., 1996,Proc. Natl. Acad. Sci. 93:2348-2352). Assay conditions (time and amountof extract used) were varied to be within the linear range of the assay.The amount of product was determined by spectrophotometry andβ-galactosidase enzyme activity was calculated. The Coomassie Plusprotein assay (Pierce) was used to determine the protein concentrationof the extracts, and results were expressed as units of β-galactosidasenormalized to total protein content of the extract. The amount ofbackground activity from the mock-infected cells was subtracted from thetotal amount of enzyme activity for each of the promoters. Eachinfection was performed in triplicate, and expressed as the mean averagewith standard deviation (Table 6).

As shown in Table 6, the incorporation of viral or mammalian cellularpromoters into baculoviruses allows for expression of an exogenous geneproduct in mammalian cells. The CMV promoter led to the highest level ofβ-galactosidase activity, with the RSV and β-actin promoters producinglower levels of β-galactosidase activity. At the moi of virus employedin this example, the albumin and PGK promoters showed no activity abovebackground levels in extracts of cells that were not treated withbutyrate, although positively stained cells were detected by X-galstaining. The addition of sodium butyrate to the cells after infectionled to detectable levels of β-galactosidase expression with all of thepromoters tested. After treating cells with sodium butyrate, the CMVpromoter showed a five-fold increase in expression of theβ-galactosidase reporter gene. The RSV LTR, albumin, pGK1, and β-actinpromoters all led to increased gene expression after treatment withbutyrate. Without being bound to any particular theory, it is postulatedthat sodium butyrate increases cellular differentiation and histoneacetylation, which increases transcription.

TABLE 6 COMPARISON OF VARIOUS PROMOTER STRENGTHS WITH AND WITHOUT SODIUMBUTYRATE Hep G2 Hep G2 Rat Hepatocytes Promoter −butyrate +butyrate−butyrate CMV  17 ± 1.4^(a) 86 ± 33  18 ± 1.2 RSV 1.0 ± 0.1 2.2 ± 0.10.25 ± 0.11 pGK1 0.0 ± 0.0 0.02 ± 0.02 0.64 ± 0.58 Albumin 0.0 ± 0.00.08 ± 0.04 0.15 ± 0.08 β-actin  0.1 ± 0.01 0.05 ± 0.02 0.25 ± 0.07^(a)Promoter strength is expressed in Units/mg of β-galactosidase.

Analysis of RNA Expression From Viral Promoters in HeDG2 Cells: Oneadvantage of using a non-mammalian virus to express an exogenous gene ina mammalian cell is that, due to a lack of appropriate host cellfactors, the non-mammalian viral promoters may not be active in themammalian cell. To determine whether AcMNPV viral gene are expressed inHepG2 cells, the viral RNA was analyzed. In these experiments, HepG2cells were infected with the Z4 virus at a moi of approximately 30. At18 hours post-infection, the cells were harvested, and total cellularRNA was extracted from the cells. The total cellular RNA was analyzed byNorthern blotting for expression of viral genes. The probe included a1.7 kbp PacI-SalI fragment from pAcUW1 (Pharmingen) which contains theviral late gene, p74, as well as the very late (hyperexpressed) gene,p10. Total cellular RNA from Z4-infected Sf9 insect cells was employedas a positive control. While extremely strong signals were detected forp10 and p74 for the control insect cells, no signal was observed forZ4-infected HepG2 cells or uninfected control cells.

Additional experiments that used reverse transcriptase-PCR (RT-PCR), ahighly sensitive method, provided further evidence that the majority ofviral v genes are not transcribed in the mammalian HepG2 cells. RT-PCRanalysis was performed with RNA prepared from Z4-infected HepG2,uninfected HepG2, or infected Sf9 cells at 6 or 24 hours post-infection.HepG2 cells were infected at a moi of 10 or 100. At 6 hourspost-infection, no RT-PCR product was observed from the viral p39, ETL,LEF1, IE1, or IE-N genes at either dose of virus in Z4-infected HepG2cells. In contrast, RT-PCR products were readily detected in Z4-infectedSf9 cells. At 24 hours post-infection, no expression of these gene wasdetected in HepG2 cells infected at a moi of 10. At 24 hourspost-infection, no expression of the viral p39, ETL, or LEF1 genes wasobserved in HepG2 cells infected at an moi of 100. However, at this highdoes of virus, low levels of expression from the viral IE1 and IE-Ngenes was observed. The low level of expression detected at an moi of100 was nonetheless significantly lower than the level of expression ininsect cells.

Expression of these genes may result from recognition of the viral TATAbox by mammalian transcription factors (i.e., transcription of theimmediate early genes by RNA polymerase II (see, e.g., Hoopes andRorhman, 1991, Proc. Natl. Acad. Sci. 88:4513-4517). In contrast to theimmediate early genes, the late or very late viral genes are transcribedby a virally-encoded RNA polymerase that, instead of requiring a TATAbox, initiates transcription at a TAAG motif (O'Reilly et al., supra).Accordingly, expression of the viral late or very late genes isnaturally blocked in mammalian cells. If desired, expression of theimmediate early genes can be blocked by deleting those genes, usingconventional methods.

While certain viruses have an intrinsic ability to infect liver cells,infection of liver cells by other viruses may be facilitated by acellular receptor, such as a cell-surface asialoglycoprotein receptor(ASGP-R). HepG2 cells differ from Sk-Hep-1 human hepatocytes and NIH3T3mouse fibroblast cells by the presence of ASGP-R on the cell surface. Incertain of the above experiments, β-galactosidase was expressed in fewerSk-Hep-1 cells (FIG. 14B) or NIH3T3 cells than HepG2 cells. The lacZgene was expressed in HepG2 cells at a frequency estimated as greaterthan 1,000 fold more than that in Sk-Hep-1 cells, based on quantitativecounts of X-gal stained cells. Normal hepatocytes have 100,000 to500,000 ASGP-R, with each receptor internalizing up to 200 ligands perday. The ASGP-R may facilitate entry of the virus into the cell byproviding a cell-surface receptor for glycoproteins on the virion. Theglycosylation patterns of insect and mammalian cells differ, with thecarbohydrate moieties on the surface of the virion produced in insectcells lacking terminal sialic acid. Those carbohydrate moieties maymediate internalization and trafficking of the virion. In addition tothe ASGP-R, other galactose-binding lectins that exist in mammals (see,e.g., Jung et al., 1994, J. Biochem. (Tokyo) 116:547-553) may mediateuptake of the virus.

If desired, the cell to be infected can be modified to facilitate entryof the baculovirus into the cell. For example, ASGP-R can be expressedon the surface of a cell to be infected by the virus (e.g.,baculovirus). The genes encoding the ASGP-R have been cloned (Spiess etal., 1985, J. Biol. Chem. 260:1979 and Spiess et al., 1985, Proc. Natl.Acad. Sci. 82:6465), and standard methods (e.g., retroviral,adeno-associated virus, or adenoviral vectors or chemical methods) canbe used for expression of the ASGP-R in the cell to be infected by avirus. Other suitable mammalian lectins can be substituted for theASGP-R in such methods (see, e.g., Ashwell et al., 1982, Ann. Rev.Biochem. 51:531-534). Other receptors for ligands on the virion, such asreceptors for insect carbohydrates or the CD4 receptor for HIV, can alsobe expressed on the surface of the mammalian cell to be infected tofacilitate infection (see, e.g., Monsigny et al., 1979, Biol. Cellulaire33:289-300).

Entry into the cell also can be facilitated by modifying the virion,e.g., through chemical means, to enable the virion to bind to otherreceptors on the mammalian cell (see, e.g., Neda, et al., 1991, J. Biol.Chem. 266:14143-14146 and Burns et al., 1993, Proc. Natl. Acad. Sci.90:8033-8037).

II. Therapeutic Use of a Non-mammalian DNA Virus Expressing an ExogenousGene

The discovery that a non-mammalian DNA virus efficiently expressed alacZ reporter gene in several mammalian cells indicates that anon-mammalian DNA virus can be used therapeutically to express anexogenous gene in a cell of a mammal. For example, the method of theinvention can facilitate expression of an exogenous gene in a cell of apatient for treatment of a disorder that is caused by a deficiency ingene expression. Numerous disorders are known to be caused by singlegene defects (see Table 7), and many of the genes involved in genedeficiency disorders have been identified and cloned. Using standardcloning techniques (see, e.g., Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, (1989)), a non-mammalian virus canbe engineered to express a desired exogenous gene in a mammalian cell(e.g., a human cell).

TABLE 7 EXAMPLES OF DISORDERS THAT CAN BE TREATED WITH THE INVENTION ANDGENE PRODUCTS THAT CAN BE MANUFACTURED WITH THE INVENTION Gene ProductDisorder fumarylacetoacetate hydrolase hereditary tyrosinemiaphenylalanine hydroxylase phenylketonuria LDL receptor familialhypercholesterolemia alpha-1 antitrypsin alpha-1 antitrypsin deficiencyglucose-6-phosphatase glycogen storage diseases porphobilinogendeaminase diseases caused by errors in porphyrin metabolism, e.g., acuteintermittent porphyria CPS-I, OTC, AS, ASL, or arginase disorders of theurea cycle factors VIII & IX hemophilia cystathione β-synthasehomocystinuria branched chain ketoacid maple syrup urine diseasedecarboxylase albumin hypoalbuminemia isovaleryl-CoA dehydrogenaseisovaleric acidemia propionyl CoA carboxylase propionic acidemia methylmalonyl CoA mutase methylmalonyl acidemia glutaryl CoA dehydrogenaseglutaric acidemia insulin insulin-dependent diabetes β-glucosidaseGaucher's disease pyruvate carboxylase pyruvate carboxylase deficiencyhepatic phosphorylase or glycogen storage diseases phosphorylase kinaseglycine decarboxylase, H-protein, non-ketotic hyperglycinemias orT-protein Wilson's disease copper- Wilson's disease transporting ATPaseMenkes disease copper- Menkes disease transporting ATPase cysticfibrosis transmembrane cystic fibrosis conductance regulator

The invention can also be used to facilitate the expression of a desiredgene in a cell having no obvious deficiency. For example, the inventioncan be used to express insulin in a hepatocyte of a patient in order tosupply the patient with insulin in the body. Other examples of proteinsthat can be expressed in a mammalian cell (e.g., a liver cell) fordelivery into the system circulation of the mammal include hormones,growth factors, and interferons. The invention can also be used toexpress a regulatory gene or a gene encoding a transcription factor(e.g., a VP16-tet repressor gene fusion) in a cell to control theexpression of another gene (e.g., genes that are operably-linked to atet operator sequence; see, e.g., Gossen et al., 1992, Proc. Natl. Acad.Sci. 89:5547-5551). In addition, the invention can be used in a methodof treating cancer by expressing in a cell a cancer therapeutic gene,such as a gene encoding a tumor suppressor (e.g., p53), tumor necrosisfactor, thymidine kinase, diphtheria toxin chimera, or cytosinedeaminases (see, e.g., Vile and Russell, 1994, Gene Therapy 1:88-98).

Other useful gene products include RNA molecules for use in RNA decoy,antisense, or ribozyme-based methods of inhibiting gene expression (see,e.g., Yu et al., 1994, Gene Therapy 1:13-26). If desired, the inventioncan be used to express a gene, such as cytosine deaminase, whose productwill alter the activity of a drug or prodrug, such as 5-fluorocytosine,in a cell (see, e.g., Harris et al., 1994, Gene Therapy 1: 170-175).Methods such as the use of ribozymes, antisense RNAs, transdominantrepressors, polymerase mutants, or core or surface antigen mutants canbe used to suppress hepatitis viruses (e.g., hepatitis virus A, B, C, orD) in a cell. Other disorders such as familial hemachromatosis can alsobe treated with the invention by treatment with the normal version ofthe affected gene.

Preferred genes for expression include those genes that encode proteinsthat are expressed in normal mammalian cells (e.g., hepatocytes or lungcells). For example, genes encoding enzymes involved in the urea cycle,such as the genes encoding carbamoyl phosphate synthetase (CPS-I),ornithine transcarbamylase (OTC), arginosuccinate synthetase (AS),arginosuccinate lyase (ASL), and arginase are useful in this method. Allof these genes have been cloned (for OTC, see Horwich et al., 1984,Science 224:1068-1074 and Hata et al., 1988, J. Biochem (Tokyo)103:302-308; for AS, see Bock et al., 1983, Nucl. Acids Res. 11:6505;Surh et al., 1988, Nucl. Acids Res. 16:9252; and Dennis et al., 1989,Proc. Natl. Acad. Sci. 86:7947; for ASL, see O'Brien et al., 1986, Proc.Natl. Acad. Sci. 83:7211; for CPS-I, see Adcock et al., 1984, (Abstract)Fed. Proc. 43:1726; for arginase, see Haraguchi et al., Proc. Natl.Acad. Sci. 84:412). Subcloning these genes into a baculovirus can bereadily accomplished with common techniques.

The therapeutic effectiveness of expressing an exogenous gene in a cellcan be assessed by monitoring the patient for known signs or symptoms ofa disorder. For example, amelioration of OTC deficiency and CPSdeficiency can be detected by monitoring plasma levels of ammonium ororotic acid. Similarly, plasma citrulline levels provide an indicationof AS deficiency, and ASL deficiency can be followed by monitoringplasma levels of arginosuccinate. Parameters for assessing treatmentmethods are known to those skilled in the art of medicine (see, e.g.,Maestri et al., 1991, J. Pediatrics, 119:923-928).

The non-mammalian DNA virus (e.g., baculovirus) can be formulated into apharmaceutical composition by admixture with a pharmaceuticallyacceptable non-toxic excipient or carrier (e.g., saline) foradministration to a mammal. In practicing the invention, the virus canbe prepared for use in parenteral administration (e.g., for intravenousinjection (e.g., into the portal vein)), intra-arterial injection (e.g.,into the femoral artery or hepatic artery), intraperitoneal injection,intrathecal injection, or direct injection into a tissue or organ (e.g.,intramuscular injection). In particular, the non-mammalian virus can beprepared in the form of liquid solutions or suspensions in conventionalexcipients. The virus can also be prepared for intranasal orintrabronchial administration, particularly in the form of nasal dropsor aerosols in conventional excipients. If desired, the virus can besonicated in order to minimize clumping of the virus in preparing thevirus.

In practicing the invention, the virus can be used to infect a celloutside of the mammal to be treated (e.g., a cell in a donor mammal or acell in vitro), and the infected cell then is administered to the mammalto be treated. In this method, the cell can be autologous orheterologous to the mammal to be treated. For example, an autologoushepatocyte obtained in a liver biopsy can be used (see, e.g., Grossmanet al., 1994, Nature Genetics 6:335). The cell can then be administeredto the patient by injection (e.g., into the portal vein). In such amethod, a volume of hepatocytes totaling about 1%-10% of the volume ofthe entire liver is preferred. Where the invention is used to express anexogenous gene in a liver cell, the liver cell can be delivered to thespleen, and the cell can subsequently migrate to the liver in vivo (see,e.g., Lu et al., 1995, Hepatology 21:7752-759). If desired, the virusmay be delivered to a cell by employing conventional techniques forperfusing fluids into organs, cells, or tissues (including the use ofinfusion pumps and syringes). For perfusion, the virus is generallyadministered at a titer of 1×10⁶ to 1×10¹⁰ pfu/ml (preferably 1×10⁹ to1×10¹⁰ pfu/ml) in a volume of 1 to 500 ml, over a time period of 1minute to 6 hours. If desired, multiple doses of the virus can beadministered to a patient intravenously for several days in order toincrease the level of expression as desired.

The optimal amount of virus or number of infected cells to beadministered to a mammal and the frequency of administration aredependent upon factors such as the sensitivity of methods for detectingexpression of the exogenous gene, the strength of the promoter used, theseverity of the disorder to be treated, and the target cell(s) of thevirus. Generally, the virus is administered at a multiplicity ofinfection of about 0.1 to 1,000; preferably, the multiplicity ofinfection is about 5 to 100; more preferably, the multiplicity ofinfection is about 10 to 50.

III. Examples of Use of a Non-mammalian Virus to Express an ExogenousGene In Vivo

The following examples demonstrate that a non-mammalian DNA virus can beused to express an exogenous gene in a cell in vivo. These examples alsodemonstrate that in vivo gene expression can be achieved byadministering the virus by intravenous injection, intranasaladministration, or direct injection of the virus into the targetedtissue. The first example demonstrates expression of an exogenous genein brain cells in vivo. The second example provides evidence ofexpression of an exogenous gene in liver, following intravenousinjection of the virus. In the third example, expression of theexogenous gene is detected in skin after topical application of the Z4virus to injured skin. In the remaining examples, a virus carrying anexogenous gene was injected directly into an organ. These examplesdemonstrate in vivo expression of an exogenous gene in skin, liver,spleen, kidney, stomach, skeletal muscle, uterus, and pancreas.

Injection Into Portal Vein: For the first example, 0.5 ml of Z4 virus(≈1.4×10⁹ pfu/ml) was injected (at a rate of ≈1 ml/min) into the portalvein of a single rat. At approximately 72 hours after infection, lacZexpression was detectable in at least one liver cell of the cryosectionsthat were examined by conventional histochemical methods. The efficiencyof expression may be increased by any one, or a combination of, thefollowing procedures: (1) pre-treating the animal with growth factors;(2) partial hepatectomy, (3) administration of immunosuppressants tosuppress any immune response to the virus; (4) use of a higher titer ordose of the virus; (5) infusion of the virus by surgical perfusion tothe liver (e.g., in order to limit possible non-specific binding of thevirus to red blood cells); and/or (6) sonication of the virus tominimize clumping of the virus.

Expression in Brain: For the second example, a 2 μl sample of Z4 virus(at a titer of 4.8×10¹ pfu/ml) was injected, using stereotacticprocedure, into the olfactory bulb in the brain of an anesthetized adultrat. The virus was injected slowly (over a 30 minute time period) toavoid compressing the brain tissue. At 1 day post-injection, the rat waseuthanized, and the brain tissue was processed for i detection ofexpression of the exogenous lacZ gene by X-gal histochemistry. Injectionof the Z4 virus into the brain resulted in in vivo expression of lacZ,as was evidenced by patches of cells that were strongly stained blue.More than 10⁴ cells were stained blue upon injection of approximately10⁷ pfu. These data thus indicate that an exogenous gene can beexpressed in the brain of a mammal by injecting into the brain anon-mammalian DNA virus whose genome includes the exogenous gene.

Topical Application and Expression in Skin: This example demonstratesthat topical application of the Z4 virus to abraded skin of a mouse canresult in expression of a heterologous gene in the skin. Theseexperiments involved four differently-treated areas on the skin of amouse. Two of the areas (an abraded and a non-abraded area) were treatedwith phosphate-buffered saline. The other two areas (an abraded and anon-abraded area) were treated with the Z4 virus (50 μl at 4.8×10¹pfu/ml). After treatment, each area of the skin was cut into sectionsusing a cryostat.

Topical application of the Z4 virus (50 μl at 4.8×10¹⁰ pfu/ml) toinjured skin of a mouse resulted in expression of the exogenous gene innearly 100% of the cells of the basal layer of the epidermis. Stainingof deeper structures was not detected. In one cryostat section, variousareas of the epidermis were stained in multiple sections. In a secondcryostat section, occasional blue cells were present. In a thirdcryostat section, patches of staining were detected, and in a fourthcryostat section, the staining was nearly continuous and very dark.Although the pattern of gene expression varied slightly between the fourcryostat sections obtained from this area of skin, the exampledemonstrates that topical application of the Z4 virus to abraded skinconsistently resulted in expression of the heterologous gene in skin.

Injection Into a Tissue or Organ: In the following examples, expressionof an exogenous gene was detected in vivo after a non-mammalian DNAvirus carrying the gene was injected directly into four distinct organs.For these examples, the Z4 virus was prepared from 1 L of Z4-infected(moi of 0.5) Sf9 cells grown in spinner culture in serum-free medium.The cells and debris were removed by centrifuging the cell culture at2000 rpm for 10 minutes. The virus was pelleted by centrifugationthrough a sucrose cushion in an SW28 rotor at 24,000 rpm for 75 minutes.For preparation of this virus stock, 33 ml of cleared virus was layeredover a 3 ml sucrose cushion (27% sucrose (w/v) in 10 mM Tris-HCl (pH7.5), 1 mM EDTA (TE)). The virus was resuspended by overnight incubationat 4° C. in 0.3 ml TE per tube. The virus was purified by banding in a20-60% sucrose (w/v in TE) gradient in SW41 tubes that were centrifugedat 38,000 rpm for 75 minutes. The virus bands were collected with asyringe and pelleted in SW50.1 rotor centrifuged at 30,000 rpm for 60minutes. The virus pellet was resuspended in a total of 0.7 ml PBS byovernight incubation at 4° C. The titer of the concentrated Z4 stock, asdetermined in a conventional plaque assay, was 4.8×10¹⁰ pfu/ml.

To assay for gene expression in vivo, the Z4 virus was administeredBalb/c female mice by direct injection of a 50 μl aliquot of theconcentrated virus (2.4×10⁹ pfu total) into either the liver, spleen,kidney, muscle, uterus, pancreas, or skin of a mouse. Surgery wasrequired for administration to liver, spleen and kidney. To spread thevirus throughout 4n organ, the 50 μl virus sample was injected into twoor three sites in an organ. A 50 μl sample of PBS was used as a negativecontrol. For assaying gene expression in the liver, only one lobe of theliver was injected, and a separate mouse received the PBS injection as anegative control. For assaying gene expression in the spleen, anuninjected mouse served as a negative control. For assaying geneexpression in kidney, muscle, and skin, contralateral controls wereperformed (the Z4 virus was injected into the right side of the organ,and PBS was injected into the left of the organ). For assayingexpression in muscle, the virus was injected into the tibealis anteriorhind leg muscle after shaving the mouse. For assaying expression inskin, the abdomen of the mouse was shaved, and 50 μl of Z4 virus wereinjected into a marked section of the abdomen. At 24 hourspost-injection, the mice were sacrificed and dissected. The Z4- andPBS-injected organs were frozen in liquid nitrogen, and 7 μm thinsections were prepared using a cryostat (Reichert-Jung Cryocut 1800).β-galactosidase activity was measured by fixing the thin sections andstaining with X-gal, as described above. Each of the organs thatreceived the Z4 virus expressed the exogenous lacZ gene in vivo. In eachcase, the PBS negative control did not promote expression of theexogenous gene.

Injection and Expression in Skin: In this example, in vivo expression ofthe exogenous lacZ gene of Z4 was observed in mouse skin after injectionof 2.4×10⁹ pfu into the skin. A high level of expression (over 25% ofcells within the area of injection) was achieved in the dermis aftersubcutaneous injection of the virus. Although the muscle layer waspredominantly unstained, positive staining of some skeletal musclefibers was observed. As a negative control, PBS was injected into theskin. Although some staining was observed in the sebaceous glands, it ismost probably due to the presence of bacteria. A low level of stainingwas also detected in the dermis. Similar results were obtained when theZ4 virus was applied topically to uninjured (non-abraded) skin, althoughno clear epidermal staining was detected. Nonetheless, these dataindicate that the Z4 virus can be used to express a heterologous gene inthe skin of a mammal when the virus is injected subcutaneously into themammal.

Expression in Liver: In this example, expression of the exogenous genewas detected in liver. Blue coloration, indicative of β-galactosidaseexpression, was detected in multiple areas of the injected lobe.Although the most intense coloration was at the point of injection, theinternal areas of the liver sections exhibited the blue coloration thatis indicative of gene expression. Expression of the exogenous geneappeared to be detected both in hepatocytes and Kupffer cells of thelobes that received the Z4 virus. In contrast, uninjected lobes from thesame liver were negative. These results thus indicate that an exogenousgene can be expressed in a liver cell by injecting into the liver anon-mammalian DNA virus encoding the gene.

Expression in Spleen: In this example, thin sections of the spleen wereassayed for gene expression following injection of the virus carryingthe exogenous gene into the spleen. Spleen cells that had received theZ4 virus in vivo expressed the lacZ gene. The blue coloration wasdetected in cells located throughout the entire spleen. The intensity ofblue coloration obtained with spleen cells was less than the intensityobtained with liver cells. Nonetheless, the blue coloration wasindicative of significant expression of the exogenous gene. No bluecoloration was detected in a spleen that did not receive the virus.These data thus indicate that an exogenous gene can be expressed in aspleen cell in vivo upon injection of a non-mammalian DNA virus whosegenome carries the gene.

Expression in Kidney: In this example, in vivo expression of anexogenous gene was detected in a kidney that was injected with Z4 asdescribed above. The Z4-injected kidney displayed clear blue coloringthat is indicative of lacZ expression; in contrast, a PBS-injectedcontrol kidney displayed no blue coloration. The blue coloration wasprimarily around the edges of the sections of the kidney. Indirectimmunofluorescence also indicated that the viral particles wereconcentrated in the edges of the sections, providing a correlationbetween gene expression and localization of the virus. These data thusindicate that a non-mammalian DNA virus can be used to express anexogenous gene in a kidney cell in vivo.

Expression in Stomach: In this example, the Z4 virus (50 μl) wasinjected into the center of the stomach of Balb/C mice. The animals weresacrificed on the day following injection, and the stomachs were frozenin liquid nitrogen, and cryostat sectioned and stated as previouslydescribed. Cell transfection was observed in gastric mucosal and musclecells. Positive staining was detected in glands, with most stainingoccurring at the bases of the glands. These observations indicate that anon-mammalian DNA virus can be used to express a heterologous gene inthe stomach of mice. In these experiments, blue staining was alsodetected in the lumen. The blue coloration in that particular region mayresult from bacteria in the gut, rather than expression from the virus.

Expression in Skeletal Muscle: In this example, in vivo expression ofthe exogenous lacZ gene of Z4 was detected in muscle after directinjection of virus into the tibialis anterior. Blue coloration was foundonly in discrete loci in the muscle, and the coloration was not asintense or widespread as the coloration observed in liver, spleen, orskin. Nonetheless, the blue coloration was significant, indicating thata non-mammalian DNA virus can be used to express an exogenous gene inmuscle in vivo.

Expression in Uterus: In this example, expression of the lacZ reportergene was detected in the uterus. A 50 μl aliquot of the Z4 virus(2.4×10⁹ pfu) was injected directly into the uterus of a mouse. Theanimal was sacrificed on the day following injection, and cryostatsections were prepared as previously described. Staining of the sectionswith X-gal produced blue coloration in an area of the uterus with littletissue disruption. The positive cells were mostly endometrial stromalcells, rather than gland elements. These data indicate that anon-mammalian DNA virus can be used to express a heterologous gene inthe uterus of a mammal.

Expression in Pancreas: This example demonstrates that a non-mammalianDNA virus can be used to express a heterologous gene in the pancreas ofa mammal. A 50 μl aliquot of the Z4 virus (2.4×10⁹ pfu) was injecteddirectly into the pancreas of a mouse. On the day following injection,the mouse was sacrificed, and the pancreas was stained with X-galaccording to conventional methods. Large areas of positive cells weredetected, indicating that the Z4 virus successfully expressed the lacZgene in the pancreas.

Summary: In sum, these examples demonstrate that a non-mammalian DNAvirus (e.g., a baculovirus) can be used to express an exogenous gene ina mammalian cell in vivo. These examples employed several distinctanimal model systems and methods of administering the virus. In each andevery case, the non-mammalian DNA virus successfully expressed theexogenous gene in vivo. These data thus provide support for theassertion that a non-mammalian DNA virus can be used to express anexogenous gene in other, non-exemplified cells in vivo. In addition, inat least some tissues, the level of expression in vivo was,surprisingly, higher than the level that would have been predicted fromthe corresponding in vitro experiments (e.g., the brain versus culturedneurons). All of these examples provide evidence of the in vivo utilityof the invention.

Part B: An Altered Coat Protein Enhances the Ability of a Non-MammalianDNA Virus to Express an Exogenous Gene in a Mammalian Cell

Now that it has been demonstrated that a non-mammalian DNA virus can beused to express an exogenous gene in a mammalian cell, the followingexamples are provided to demonstrate that an altered coat proteinenhances the ability of the non-mammalian DNA virus to express theexogenous gene in a mammalian cell.

Construction of Baculovirus Transfer Plasmid pVGZ3

These examples employ a baculovirus that has been engineered to expressa vesicular stomatitis virus glycoprotein G (VSV-G) as an altered coatprotein. The baculovirus transfer plasmid pVGZ3 is a derivative of thebaculovirus transfer plasmid Z4, which was used in many of the examplessummarized above.

The baculovirus transfer plasmid pVGZ3 was constructed by insertingexpression cassettes encoding VSV-G and the reporter gene lacZ into thebaculovirus transfer vector BacPAK9 (Clontech; Palo Alto, Calif.). Toproduce this transfer plasmid, cDNA encoding VSV-G was excised as a1,665 bp BamHI fragment from pLGRNL (Burns, 1993, Proc. Natl. Acad. Sci.90:8033-8037). The resulting plasmid was termed VSVG/BP9 (FIG. 18). TheRSV LTR, lacZ gene, and SV40 polyadenylation signal were excised fromthe Z4 transfer plasmid using BglII and BamHI to produce a 4,672 bpfragment. This fragment was inserted into the BglII site of VSVG/BP9such that the lacZ gene was positioned downstream from the VSV-G gene toproduce the VGZ3 transfer plasmid (FIG. 19). In VGZ3, the directions oftranscription of the VSV-G and lacZ genes are convergent. In otherwords, the promoters lie at opposite ends of the inserted sequences,with the lacZ and VSV-G genes being transcribed towards each other. TheSV40 polyA site is bidirectional and used by both the VSV-G and lacZgenes.

Because the VSV-G gene in this transfer plasmid is operably linked to abaculovirus polyhedrin promoter, the plasmid offers the advantage ofhigh levels of expression of the VSV-G gene in insect cells andrelatively low levels of expression in mammalian cells. This pattern ofexpression of the altered coat protein is desirable because the alteredcoat protein is produced efficiently in insect cells, where thenon-mammalian DNA virus having an protein is manufactured before thevirus is delivered to a mammalian cell. Producing the virus in insectcells before using the virus to infect mammalian cells obviates concernsabout expressing a viral coat protein (e.g., VSV-G) in mammalian cells.

A schematic representation of a budding virus having an altered coatprotein is provided in FIG. 20. Once the virus infects a mammalian cell,expression of the altered coat protein is not desirable. Therefore, theuse of an non-mammalian (e.g., insect) virus promoter, which is notactive in mammalian cells, is preferable for driving expression of thealtered coat protein. By contrast, the exogenous gene of interest (here,the lacZ reporter gene) is operably linked to an RSV LTR. As is desired,expression of a gene driven by the RSV LTR is obtained both in insectcells and in mammalian cells.

Standard procedures were used to produce the recombinant baculovirusVGZ3 from the pVGZ3 transfer plasmid, described above. Briefly, Sf9cells were co-transfected, according to the manufacturer's instructions,by lipofection with pVGZ3 and the baculovirus genomic DNA BacPAK6(Clontech; Palo Alto, Calif.) that had been digested with Bsu36I. Thevirus was plaque purified and amplified according to standardtechniques. Using a conventional plaque assay on Sf9 cells, the viraltiter was determined to be 3.1×10⁷ pfu/ml.

Enhanced Expression in HepG2, Vero, and HeLa Cells: This exampledemonstrates that VGZ3, the baculovirus having an altered coat protein,has an enhanced ability, relative to the Z4 virus described above, toexpress an exogenous gene in a mammalian cell. To provide sensitivity inthe assays, these experiments were performed under conditions in whichthe Z4 virus expresses an exogenous gene at relatively low levels. Threedifferent cell types were used, including HepG2 cells, Vero cells (akidney cell line), and HeLa cells (a cervical carcinoma cell line). Inthese experiments, 1×10⁵ cells of each cell type were, independently,seeded into multiple wells of 12-well culture plates. On the followingday, the tissue culture medium was replaced with fresh medium, and theZ4 and VGZ3 viruses were added, separately, to the cells. The viruseswere used at multiplicities of infection of 0, 1.25, 10, and 80(assuming that the cell number had doubled overnight), and eachexperiment was performed in duplicate. On the day following the additionof virus, the cells were harvested, and expression of the exogenous lacZgene was detected by X-gal staining or by using a quantitativechemiluminescent β-galactosidase assay (Clontech; Palo Alto, Calif.).The results of these assays are presented in Table 8.

TABLE 8 USE OF A NON-MAMMALIAN DNA VIRUS HAVING AN ALTERED COAT PROTEINTO ACHIEVE ENHANCED EXPRESSION OF AN EXOGENOUS GENE IN HEPG2 AND HELACELLS Chemiluminescence VGZ3 Cell Line Virus moi Units Superiority^(a)HepG2 Z4 80 16.4 HepG2 VGZ3 80 180.2 11.0-fold HeLa Z4 80 0.02^(b) HeLaVGZ3 80 1.75 >87.5-fold HeLa VGZ3 1.25 0.07 >224 fold^(c)^(a)Superiority was calculated as VGZ3 transduction units ÷ Z4transduction units for each cell type. ^(b)0.02 was the background levelin the chemiluminescent assay. ^(c)The difference in moi (1.25 for VGZ3and 80 for Z4) was accounted for in determining the VGZ3 superiority.

This example demonstrates that a baculovirus that is engineered toexpress a VSV glycoprotein G has an enhanced ability, relative to abaculovirus that lacks the altered coat protein, to express an exogenousgene in a mammalian cell. In this example, expression of the exogenouslacZ gene was detected in 100% of the HepG2 cells that were contactedwith VGZ3 at an moi of 80. In contrast, under these conditions,expression of the exogenous gene was detected in only 15% of the cellsthat were contacted with the Z4 virus (a virus that does not have analtered coat protein). Accordingly, these data show that altering a coatprotein enhances the ability of a virus to express an exogenous gene ina mammalian cell.

Enhanced expression of the exogenous gene was also achieved with Verocells: over 50% of the VGZ3-treated cells turned blue, whereas only5-10% of the Z4-treated cells turned blue upon staining with X-gal.Further evidence that the altered coat protein enhances the ability of avirus to express and exogenous gene in a mammalian cell comes from arelatively sensitive assay employing HeLa cells. Under the conditionsemployed in this example, the Z4 virus does not efficiently express anexogenous gene in HeLa cells. No blue cells were detected at an moi of80, indicating that the frequency of gene expression was less than1×10⁵. By contrast, approximately 3% of the HeLa cells treated with theVGZ3 virus were blue, indicating that the efficiency of gene expressionwas 3×10⁻², which is 12,000-fold better than the efficiency obtainedwith Z4. With the VGZ3 virus, blue HeLa cells were also detected at thelow moi of 1.25, whereas no blue cells were detected at the moi with theZ4 virus.

In sum, these data indicate that an altered coat protein on anon-mammalian DNA virus can enhance the ability of that virus to infectand express a gene in a mammalian cell. In addition, employment of analtered coat protein allows the virus to infect a cell at a lower moi.Thus certain cells that appear to be refractive to the virus at a givenmoi (e.g., HeLa cells) can be infected with a virus having an alteredcoat protein, thereby expanding the apparent host range of the virus. Avirus having an altered coat protein thus offers the advantage ofpermitting expression of an exogenous gene at a low moi, relative to themoi needed with a virus that lacks an altered coat protein.

As was described above for the Z4 virus, exogenous gene expression incells treated with the VGZ3 virus results from de novo gene expressionwithin the mammalian cell. Both cycloheximide, which inhibits proteinsynthesis, and chloroquine, which inhibits endosome acidification,separately inhibited β-galactosidase expression in VGZ3-treatedmammalian cells (data not shown). Thus, β-galactosidase detected in themammalian cells in these experiments can be attributed to de novoprotein synthesis within the mammalian cell.

Enhanced Expression in PC12 Cells: In this example, the VGZ3 virus isshown to increase the level of exogenous gene expression in rat corticalneuronal cells, relative to the level obtained with the Z4 virus. Usingconventional methods, PC12 cells were plated at 10,000 cells/well in a24-well dish. On day 1, the cell culture medium (DMEM containing 5% FBSand 10% horse serum) was replaced with fresh cell culture medium thatalso contained 50 ng/ml of nerve growth factor (NGF). FreshNGF-containing-medium was again added at days 3 and 5. On day 6, thecells were infected with various dilutions of Z4 virus and VGZ3 virus,as shown in Table 9. At day 7, the cells were fixed and stained forβ-galactosidase by immunocytochemistry using an anti-β-galactosidaseantibody (available from 5′→3′ Inc.). Under these conditions, fewer than1% of the PC12 cells infected with 2×10⁸ pfu of Z4 expressed theexogenous gene, and approximately 17.5% of the cells infected with 1×10⁷pfu of VGZ3 expressed the exogenous gene. Accordingly, these dataprovide additional evidence that a virus having an altered coat proteinhas a superior ability to express an exogenous gene in a mammalian cell.

Enhanced Expression in Primary Rat Cortical Cells

This example demonstrates that a virus having an altered coat proteinprovides enhanced expression of an exogenous gene in a primary culturesof rat cortical cells, as compared with a virus lacking the altered coatprotein. For this example, cultures of rat cortical cells were preparedand infected as described above under “Expression in Cortex Cultures.”The Z4 and VGZ3 viruses were used at the moi shown in Table 9 (note: themoi of Z4 was approximately 10-fold higher than the moi of VGZ3). Whenthe number of blue cells obtained is compared with the moi of Z4 or VGZ3used, it becomes apparent that the VGZ3 virus is more efficient atexpressing the exogenous gene in the mammalian cells than is the Z4virus. In addition, this example demonstrates that a non-mammalian DNAvirus having an altered coat protein can direct exogenous geneexpression in primary rat neurons (in addition to the cell line PC12, asshown above).

TABLE 9 USE OF A NON-MAMMALIAN DNA VIRUS HAVING AN ALTERED COAT PROTEINTO ACHIEVE ENHANCED EXPRESSION OF AN EXOGENOUS GENE IN PRIMARY CULTURESOF RAT CORTICAL CELLS Virus 1 μl 2 μl 5 μl 10 μl 50 μl 100 μl Z4 moi =moi = 2 moi = 5 moi = moi = moi = 1^(a) 10 50 100 no blue no blue ≈5blue ≈20 ≈500 ≈2200 cells cells cells blue blue blue cells cells cells≈0.75% VGZ3 moi = moi = moi = moi = 1 moi = 5 moi = 10 0.1 0.2 0.5 noblue no blue no blue ≈10 ≈200 ≈60 cells cells cells blue blue blue cellscells cells^(b) PBS no blue no blue no blue cells cells cells ^(a)Themoi's were estimated based on the number of cells plated the day beforeinfection. ^(b)Because of cell death occurring in this well, fewerstained cells were detected. Nonetheless, the percentage of blue cellswas high.

Enhanced Expression in HepG2, HuH7, HeLa, WISH, A549, VERO, CHO, andBalb/c 3T3 Cells: Further data showing that an altered coat proteinenhances the ability of a non-mammalian DNA virus to direct expressionof an exogenous gene in mammalian cells is provided by this example.Here, a variety of cells were infected with the VGZ3 baculovirus. Themethods employed in these experiments first are described.

Cells: The human hepatoma lines HepG2 and HuH7, the human cervicalcarcinoma line HeLa, the human amniotic cell line WISH, the human lungcarcinoma A549, the African green monkey kidney line VERO, the hamsterepithelial line CHO and the mouse embryonic fibroblast line Balb/c 3T3were all obtained from ATCC. All mammalian cells were grown inDulbecco's Modified Eagle's Medium (GibcoBRL, Grand Island, N.Y.) with10% fetal bovine serum and 4 mM glutamine (BioWhittaker, Walkersville,Md.), except for WISH cells, which were grown in MEM with Hanks's salts(GibcoBRL), 20% fetal bovine serum and 4 mM glutamine.

Infection and Reporter Gene Assay: Cells were seeded at 2×10⁵ cells perwell in 12-well plates. After the cells attached to the plastic, thecells were rinsed with medium and fresh complete medium was added. Viralinfection was performed by adding virus to the medium at the indicatedmultiplicities of infection (moi). Following an 18-24 hour incubation at37° C. in 5% C02, cells were stained with X-gal to visualizeβ-galactosidase-expressing cells or cell lysates were taken andβ-galactosidase activity quantitated by a luminescent β-galactosidaseassay (Clontech catalog # K2048-1) according to the manufacturer'sinstructions.

Results: The use of the VGZ3 virus enhances exogenous gene expression,as compared with the level of gene expression obtained with the Z4virus. X-gal staining of infected cells in culture indicated that anapproximately 10-fold higher percentage of HepG2 cells expressed theexogenous gene following infection with VGZ3, as compared with the Z4virus (data not shown). In addition, the intensity of the blue staininghas greater in the VGZ3-treated cells, suggesting that a higher level ofgene expression within the VGZ3-infected cells. Enhanced gene expressionwas also detected when the VGZ3 virus was used to infect HeLa cells. Atan moi of 100, the Z4 virus produced few blue cells per well(approximately 1-5 cells), while approximately 10% of the VGZ3 cellsstained blue with X-gal.

The results of a quantitative assay of β-galactosidase expression areshown in FIG. 22. At each moi tested, the level of β-galactosidaseexpression in HepG2 cells treated with VGZ3 was roughly 10-fold higherthan the level obtained with the Z4 virus. The difference intransduction efficiency between the Z4 virus and the VGZ3 virus was evenmore notable in HeLa cells. At an moi of 1 or 10, no β-galactosidaseactivity above the background levels was detected with the Z4 virus. Incontrast, β-galactosidase activity was detectable in HeLa cells treatedwith VGZ3 at an moi of 1. When the Z4 virus was used at an moi of 100,β-galactosidase activity just above background levels was detected. Whenthe VGZ3 virus was used at an moi of 100, the level of β-galactosidaseactivity detected in HeLa cells was approximately 350 times greater thanthe level detected in Z4-treated cells.

A panel of 8 different cell lines was used to compare the transductionefficiencies of the Z4 and VGZ3 viruses at an moi of 50. At this lowmoi, exogenous gene expression is not detected in certain of the celllines treated with the Z4 virus, as shown in Table 10. In contrast, theVGZ3 virus led to detectable levels of exogenous gene expression in allof the cell lines at an moi of 50. In sum, these data provide furtherevidence that an altered coat protein enhances exogenous gene expressionfrom a non-mammalian DNA virus.

TABLE 10 B-GALACTOSIDASE ACTIVITY IN Z4- AND VGZ3-TREATED CELLSβ-galactosidase activity^(a) Cells Z4-treated^(b) VGZ3-treated HepG26.62 58.21 HuH7 4.46 42.49 HeLa 0.05 2.67 WISH 0.00 1.85 A549 0.22 46.34VERO 0.58 6.38 CHO 0.00 2.33 3T3 0.02 2.01 ^(a)For each cell line, theβ-galactosidase activity in uninfected cells was determined, and thisvalue was subtracted from the raw numbers for β-galactosidase activityin Z4-treated and VGZ3-treated cells. Each data point represents theaverage of three samples. ^(b)All Z4 and VGZ3 treatments were at an moiof 50.

Part C: Production of Complement-resistant Non-mammalian DNA Viruses

The following examples illustrate the production of complement-resistantnon-mammalian DNA viruses. In these examples, the non-mammalian DNAvirus was a baculovirus containing a lacZ reporter gene under thetranscriptional control of a CMV IE1 promoter. Briefly, the virus thatwas engineered to be resistant to complement was incubated with serumcontaining complement and shown to be complement-resistant, relative tovirus that was not engineered to be complement-resistant.

Example 1, illustrated by FIG. 24, shows that virus propagated on Ea4cells is more resistant to complement than is virus propagated on Sf21cells. In this experiment, 1-10 μl of virus (10⁹-10¹⁰ pfu/ml) propagatedon either Sf21 cells or Ea4 cells, as described above, was mixed withvarious concentrations (5%, 10%, 25%, and 50%) of rat complement serum(Sigma; St. Louis, Mo.), which is serum that contains complement. Eachreaction was performed in triplicate. The virus and rat complement serumwere combined at 4° C. with EMEM in a total volume of 100 μl. Themixture was incubated at 37° C. for 30 minutes then used to infect a 60mM dish of HepG2 cells containing 1 ml of serum-free EMEM. Any virusremaining in the sample after treatment with complement was allowed toattach to the HepG2 cells for 2 hours. The unattached virus then wasremoved from the cells, and the cells were re-fed with EMEM plus 10%cosmic calf serum (Hyclone). After 12-36 hours, the cells were harvestedby rinsing them with PBS, scraping the dishes, and pelleting the cells.The cells then were lysed by freezing then thawing them three times. Thelysed cells were centrifuged to pellet cellular debris, and thesupernatant was removed and used in an ELISA to detect β-galactosidaseexpressed from the lacZ reporter gene. As a control, the virus also wastreated with heat-inactivated rat complement serum, which provides 0%inhibition by complement.

As shown in FIG. 24, the virus propagated on Ea4 cells resulted in alower percent inhibition by complement than did the virus propagated onSf21 cells. In other words, the virus that was propagated on Ea4 cellswas more resistant to complement than was the virus that was propagatedon Sf21 cells.

Examples 2 and 3, illustrated by FIG. 25, demonstrate thatcomplement-resistant virus can be produced by (i) propagating the viruson cells that express galactosyltransferase or (ii) propagating a virusengineered to express siayltransferase on cells that expressgalactosyltransferase.

In example 2, Sf21 cells were engineered to express bovine β-1,4galactosyltransferase under the control of a baculovirus IE1 promoter.These cells were infected with a baculovirus containing a lacZ geneunder the control of a CMV promoter, which was propagated in the cellsas described above. The virus then was incubated with varyingconcentrations of rat complement serum, as described above, and thecomplement-treated virus was used to infect HepG2 cells. LacZ expressionin the HepG2 cells was measured by ELISA, as described above.

In example 3, Sf21 cells expressing galactosyltransferase (as describedabove) were infected with a baculovirus that expressed (a) α-2,6siayltransferase under the control of a baculoviral IE1 promoter and (b)lacZ under the control of a CMV IE1 promoter. After propagating thismodified virus on cells expressing galactosyltransferase, the virus wasincubated with varying concentrations of rat complement serum, asdescribed above. The complement-treated virus then was used to infectHepG2 cells, and lacZ expression was measured by ELISA, as describedabove.

As a control, baculovirus expressing lacZ under the control of a CMVpromoter was propagated on Sf21 cells, treated with complement, thenused to infect HepG2 cells, as described above. The results of theseexperiments are presented in FIG. 25, which shows that, relative tovirus propagated on control Sf21 cells, the virus propagated on cellsexpressing galactosyltransferase, or expressing galactosyltransferaseand infected with a virus expressing siayltransferase, resulted in alower percent inhibition by complement than did the virus propagated onSf21 cells. In other words, the virus that was propagated on the cellsexpressing galactosyltransferase (example 2), and the virus engineeredto express siayltransferase and propagated on cells expressinggalactosyltransferase (example 3), were more resistant to complementthan was the virus that was propagated on Sf21 cells.

Other Embodiments

Non-mammalian viruses other than the above-described Autographacalifornica viruses can be used in the invention; such viruses arelisted in Table 1. Nuclear polyhedrosis viruses, such as multiplenucleocapsid viruses (MNPV) or single nucleocapsid viruses (SNPV), arepreferred. In particular, Choristoneura fumiferana MNPV, Mamestrabrassicae MNPV, Buzura suppressaria nuclear polyhedrosis virus, Orgyiapseudotsugata MNPV, Bombyx mori SNPV, Heliothis zea SNPV, andTrichoplusia ni SNPV can be used.

Granulosis viruses (GV), such as the following viruses, are alsoincluded among those that can be used in the invention: Cryptophlebialeucotreta GV, Plodia interpunctella GV, Trichoplusia ni GV, Pierisbrassicae GV, Artogeia rapae GV, and Cydia pomonella granulosis virus(CpGV). Also, non-occluded baculoviruses (NOB), such as Heliothis zeaNOB and Oryctes rhinoceros virus can be used.

Other insect (e.g., lepidopteran) and crustacean viruses can also beused in the invention. Further examples of useful viruses include thosethat have infect fungi (e.g., Strongwellsea magna) and spiders. Virusesthat are similar to baculoviruses have been isolated from mites,Crustacea (e.g., Carcinus maenas, Callinectes sapidus, the Yellow HeadBaculovirus of penaeid shrimp, and Penaeus monodon-type baculovirus),and Coleoptera. Also useful in the invention is the Lymantria disparbaculovirus.

If desired, the virus can be engineered to facilitate targeting of thevirus to certain cell types. For example, ligands that bind to cellsurface receptors other than the ASGP-R can be expressed on the surfaceof the virion. Alternatively, the virus can be chemically modified totarget the virus to a particular receptor.

If desired, the cell to be infected can first be stimulated to bemitotically active. In culture, agents such as chloroform can be used tothis effect; in vivo, stimulation of liver cell division, for example,can be induced by partial hepatectomy (see, e.g., Wilson, et al., 1992,J. Biol. Chem. 267:11283-11489). optionally, the virus genome can beengineered to carry a herpes simplex virus thymidine kinase gene; thiswould allow cells harboring the virus genome to be killed bygancicylovir. If desired, the virus could be engineered such that it isdefective in growing on its natural non-mammalian host cell (e.g.,insect cell). Such strains of viruses could provide added safety and bepropagated on a complementing packaging line. For example, a defectivebaculovirus could be made in which an immediate early gene, such as IE1,has been deleted. This deletion can be made by targeted recombination inyeast or E. coli, and the defective virus can be replicated in insectcells in which the IE1 gene product is supplied in trans. If desired,the virus can be treated with neuraminidase to reveal additionalterminal galactose residues prior to infection (see, e.g., Morell etal., 1971, J. Biol. Chem. 246:1461-1467).

What is claimed is:
 1. A method for producing a complement-resistantnon-mammalian DNA virus, the method comprising: introducing into anEstigmene acrea cell a genome of a non-mammalian DNA virus selected fromthe group consisting of baculoviruses, entomopox viruses, anddensonucleosis viruses, wherein the genome comprises an exogenous geneoperably linked to a mammalian-active promoter; and allowing the virusto replicate in the Estigmene acrea cell, thereby producing acomplement-resistant non-mammalian DNA virus.
 2. The method of claim 1,wherein the cell is selected from the group consisting of an Ea4 celland a BTI-EaA E₁ acrea cell.
 3. The method of claim 1, wherein thegenome of the non-mammalian DNA virus further comprises a nucleic acidsequence encoding an altered coat protein.
 4. The method of claim 1,further comprising introducing the complement-resistant non-mammalianDNA virus into a mammalian cell.
 5. The method of claim 1, furthercomprising introducing the complement-resistant non-mammalian DNA virusinto a mammal.
 6. The method of claim 1, further comprising culturingthe cell in a cell culture medium comprising one or both of (i)D-mannosamine and (ii) N-acetyl-D-mannosamine.
 7. A method for producinga complement-resistant non-mammalian DNA virus, the method comprising:providing a non-mammalian cell that expresses one or both of (i) amammalian siayltransferase and (ii) a mammalian galactosyltransferase;introducing into the cell a non-mammalian DNA virus, wherein the genomeof the virus comprises an exogenous gene operably linked to amammalian-active promoter; and allowing the virus to replicate in thenon-mammalian cell, thereby producing a complement-resistantnon-mammalian DNA virus.
 8. The method of claim 7, wherein the genome ofthe non-mammalian DNA virus further comprises a nucleic acid sequenceencoding an altered coat protein.
 9. The method of claim 7, furthercomprising introducing the complement-resistant non-mammalian DNA virusinto a mammalian cell.
 10. The method of claim 7, further comprisingintroducing the complement-resistant non-mammalian DNA virus into amammal.
 11. The method of claim 7, further comprising culturing thenon-mammalian cell in a culture medium comprising one or both of (i)D-mannosamine and (ii) N-acetyl-D-mannosamine while the virus is allowedto replicate in the non-mammalian cell.
 12. A method for producing acomplement-resistant non-mammalian DNA virus, the method comprising:introducing into a non-mammalian cell a genome of a non-mammalian DNAvirus, wherein the genome of the virus comprises an exogenous geneoperably linked to a mammalian-active promoter; culturing thenon-mammalian cell in a culture medium comprising one or both of (i)D-mannosamine and (ii) N-acetyl-D-mannosamine; and allowing the virus toreplicate in the non-mammalian cell, thereby producing acomplement-resistant non-mammalian DNA virus.
 13. The method of claim12, wherein the genome of the non-mammalian DNA virus further comprisesa nucleic acid sequence encoding an altered coat protein.
 14. The methodof claim 12, further comprising introducing the complement-resistantnon-mammalian DNA virus into a mammalian cell.
 15. The method of claim12, further comprising introducing the complement-resistantnon-mammalian DNA virus into a mammal.
 16. A method for producing acomplement-resistant non-mammalian DNA virus, the method comprising:introducing into a non-mammalian cell a genome of a non-mammalian DNAvirus, wherein the genome of the virus comprises (i) an exogenous geneoperably linked to a mammalian-active promoter and (ii) one or both or(a) a mammalian siayltransferase gene and (b) a mammaliangalactosyltransferase gene, wherein the siayltransferase and/orgalactosyltransferase gene is operably linked to a promoter that isactive in the non-mammalian cell; and allowing the virus to replicate inthe non-mammalian cell, thereby producing a complement-resistantnon-mammalian DNA virus.
 17. The method of claim 16, wherein the genomeof the non-mammalian DNA virus further comprises a nucleic acid sequenceencoding an altered coat protein.
 18. The method of claim 16, furthercomprising introducing the complement-resistant non-mammalian DNA virusinto a mammalian cell.
 19. The method of claim 16, further comprisingintroducing the complement-resistant non-mammalian DNA virus into amammal.
 20. The method of claim 16, further comprising culturing thenon-mammalian cell in a culture medium comprising one or both of (i)D-mannosamine and (ii) N-acetyl-D-mannosamine while the virus is allowedto replicate in the non-mammalian cell.
 21. A method for producing acomplement-resistant non-mammalian DNA virus, the method comprising:providing a non-mammalian cell that expresses one or both of (i) a CD59,or a homolog thereof and (ii) a decay accelerating factor (DAF), or ahomolog thereof; introducing into the cell a non-mammalian DNA virus,wherein the genome of the virus comprises an exogenous gene under thecontrol of a mammalian-active promoter; and allowing the virus toreplicate in the non-mammalian cell, thereby producing acomplement-resistant non-mammalian DNA virus.
 22. The method of claim21, wherein the genome of the non-mammalian DNA virus further comprisesa nucleic acid sequence encoding an altered coat protein.
 23. The methodof claim 21, further comprising introducing the complement-resistantnon-mammalian DNA virus into a mammalian cell.
 24. The method of claim21, further comprising introducing the complement-resistantnon-mammalian DNA virus into a mammal.
 25. The method of claim 21,further comprising culturing the non-mammalian cell in a culture mediumcomprising one or both of (i) D-mannosamine and (ii)N-acetyl-D-mannosamine while the virus is allowed to replicate in thenon-mammalian cell.
 26. A method for producing a complement-resistantnon-mammalian DNA virus, the method comprising: introducing into anon-mammalian cell a genome of a non-mammalian DNA virus, wherein thegenome of the virus comprises (i) an exogenous gene operably linked to amammalian-active promoter and (ii) one or both or (a) a nucleotidesequence encoding CD59, or a homolog thereof, operably linked to apromoter that is active in the non-mammalian cell and (b) a nucleotidesequence encoding decay accelerating factor, or a homolog thereof,operably linked to is a promoter that is active in the non-mammaliancell; and allowing the virus to replicate in the non-mammalian cell,thereby producing a complement-resistant non-mammalian DNA virus. 27.The method of claim 26, wherein the genome of the non-mammalian DNAvirus further comprises a nucleic acid sequence encoding an altered coatprotein.
 28. The method of claim 26, further comprising introducing thecomplement-resistant non-mammalian DNA virus into a mammalian cell. 29.The method of claim 26, further comprising introducing thecomplement-resistant non-mammalian DNA virus into a mammal.
 30. Themethod of claim 26, further comprising culturing the non-mammalian cellin a culture medium comprising one or both of (i) D-mannosamine and (ii)N-acetyl-D-mannosamine while the virus is allowed to replicate in thenon-mammalian cell.
 31. An Estigmena acrea cell comprising a genome of anon-mammalian DNA virus selected from the group consisting ofbaculoviruses, entomopox viruses, and densonucleosis viruses, whereinthe genome comprises an exogenous gene under the control of amammalian-active promoter.
 32. The cell of claim 31, wherein the genomefurther comprises a nucleic acid sequence encoding an altered coatprotein.
 33. A non-mammalian cell comprising (i) a genome of anon-mammalian DNA virus, wherein the genome of the virus comprises anexogenous gene under the control of a mammalian-active promoter and (ii)one or both of (a) a nucleic acid sequence encoding a mammaliansiayltransferase and (b) a nucleic acid sequence encoding a mammaliangalactosyltransferase.
 34. The cell of claim 33, wherein the genome ofthe virus further comprises a nucleic acid sequence encoding an alteredcoat protein.
 35. A cell culture comprising: (i) a non-mammalian cellcontaining a genome of a non-mammalian DNA virus, wherein the genome ofthe virus comprises an exogenous gene operably linked to a mammalianpromoter; and (ii) cell culture media comprising one or both of (a)D-mannosamine and (b) N-acetyl-D-mannosamine.
 36. The cell culture ofclaim 35, wherein the genome of the virus further comprises a nucleicacid sequence encoding an altered coat protein.
 37. A nucleic acidcomprising a genome of a non-mammalian DNA virus, wherein the genome ofthe virus comprises (i) an exogenous gene under the control of amammalian-active promoter and (ii) one or both of (a) a nucleic acidsequence encoding a mammalian siayltransferase and (b) a nucleic acidsequence encoding a mammalian galactosyltransferase.
 38. The nucleicacid of claim 37, wherein the genome of the virus further comprises anucleic acid sequence encoding an altered coat protein.
 39. A cellcomprising the nucleic acid of claim
 37. 40. A nucleic acid comprising(i) a genome of a non-mammalian DNA virus, wherein the genome of thevirus comprises an exogenous gene under the control of amammalian-active promoter and (ii) one or both of (a) a nucleic acidsequence encoding CD59 or a homolog thereof and (b) a nucleic acidsequence encoding decay accelerating factor or a homolog thereof.
 41. Acell comprising the nucleic acid of claim
 40. 42. The cell of claim 40,wherein the genome of the virus further comprises a nucleic acidsequence encoding an altered coat protein.
 43. A non-mammalian DNA viruswherein the genome of the virus comprises an exogenous gene operablylinked to a mammalian-active promoter; and a coat protein of thenon-mammalian DNA virus comprises a mannose core region linked to acarbohydrate moiety selected from the group consisting of N-acetylglucosamine, galactose, and neuraminic acid.
 44. The non-mammalian DNAvirus of claim 43, further comprising an altered coat protein.
 45. Thenon-mammalian DNA virus of claim 43, wherein the virus is selected fromthe group consisting of a baculovirus, an entomopox virus, and adensonucleosis virus.